Methods for identification of sepsis-causing bacteria

ABSTRACT

The present invention provides compositions, kits and methods for rapid identification and quantification of sepsis-causing bacteria by molecular mass and base composition analysis.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. application Ser. No. 11/409,535, filed Apr. 21, 2006 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/674,118, filed Apr. 21, 2005; U.S. Provisional Application Ser. No. 60/705,631, filed Aug. 3, 2005; U.S. Provisional Application Ser. No. 60/732,539, filed Nov. 1, 2005; and U.S. Provisional Application Ser. No. 60/773,124, filed Feb. 13, 2006. This application is also a continuation-in-part of U.S. application Ser. No. 11/060,135, filed Feb. 17, 2005 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/545,425 filed Feb. 18, 2004; U.S. Provisional Application Ser. No. 60/559,754, filed Apr. 5, 2004; U.S. Provisional Application Ser. No. 60/632,862, filed Dec. 3, 2004; U.S. Provisional Application Ser. No. 60/639,068, filed Dec. 22, 2004; and U.S. Provisional Application Ser. No. 60/648,188, filed Jan. 28, 2005. This application is also a continuation-in-part of U.S. application Ser. No. 10/728,486, filed Dec. 5, 2003 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/501,926, filed Sep. 11, 2003. This application also claims the benefit under 35 USC 119(e) to U.S. Provisional Application Ser. No. 60/808,636, filed May 25, 2006. Each of the above-referenced U.S. Applications is incorporated herein by reference in its entirety. Methods disclosed in U.S. application Ser. Nos. 09/891,793, 10/156,608, 10/405,756, 10/418,514, 10/660,122, 10,660,996, 10/660,997, 10/660,998, 10/728,486, 11/060,135, and 11/073,362, are commonly owned and incorporated herein by reference in their entirety for any purpose.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support under CDC contract CI000099-01. The United States Government may have certain rights in the invention.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled DIBIS0088US3SEQ.txt, created on May 25, 2007 which is 252 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions, kits and methods for rapid identification and quantification of sepsis-causing bacteria by molecular mass and base composition analysis.

BACKGROUND OF THE INVENTION

A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al. Philos. Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.

Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect certain pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture.

A major conundrum in public health protection, biodefense, and agricultural safety and security is that these disciplines need to be able to rapidly identify and characterize infectious agents, while there is no existing technology with the breadth of function to meet this need. Currently used methods for identification of bacteria rely upon culturing the bacterium to effect isolation from other organisms and to obtain sufficient quantities of nucleic acid followed by sequencing of the nucleic acid, both processes which are time and labor intensive.

Sepsis is a severe illness caused by overwhelming infection of the bloodstream by toxin-producing bacteria. Although viruses and fungi can cause septic shock, bacteria are the most common cause. The most frequent sites of infection include lung, abdomen, urinary tract, skin/soft tissue, and the central nervous system. Symptoms of sepsis are often related to the underlying infectious process. When the infection crosses into sepsis, the resulting symptoms are tachycardia, tachypnea, fever and/or decreased urination. The immunological response that causes sepsis is a systemic inflammatory response causing widespread activation of inflammation and coagulation pathways. This may progress to dysfunction of the circulatory system and, even under optimal treatment, may result in the multiple organ dysfunction syndrome and eventually death.

Septic shock is the most common cause of mortality in hospital intensive care units. Traditionally, sepsis is diagnosed from multiple blood cultures and is thus, time consuming.

Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.

The present invention provides oligonucleotide primers and compositions and kits containing the oligonucleotide primers, which define bacterial bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify sepsis-causing bacteria at and below the species taxonomic level.

SUMMARY OF THE INVENTION

Disclosed herein are compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The primer pair is configured to generate an amplification product between 45 and 200 linked nucleotides in length. The forward primer is configured to hybridize with at least 70% complementarity to a first portion of a region defined by nucleotide residues 4182972 to 4183162 of Genbank gi number: 49175990 and the reverse primer is configured to hybridize with at least 70% complementarity to the second portion of the region. This oligonucleotide primer pair may have a forward primer that has at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1448. This oligonucleotide primer pair may have a reverse primer that has at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1461.

The forward primer or the reverse primer or both may have at least one modified nucleobase which may be a mass modified nucleobase such as 5-Iodo-C. The modified nucleobase may be a mass modifying tag or a universal nucleobase such as inosine.

The forward primer or the reverse primer or both may have at least one non-templated T residue at its 5′ end.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1448, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1461 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1448, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1464 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1451, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1464 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1450, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1463 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 309, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1458 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 309, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1460 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1445, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1458 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1447, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1460 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1447, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1460 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 309, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1459 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1446, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1458 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1452, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1467 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1452, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1465 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1453, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1466 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1449, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1462 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1444, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1457 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1454, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1468 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1455, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1469 or any percentage or fractional percentage sequence identity therebetween.

Also disclosed is an oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The forward primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1456, or any percentage or fractional percentage sequence identity therebetween and the reverse primer may have at least 70%, at least 80%, at least 90% or 100% sequence identity with SEQ ID NO: 1470 or any percentage or fractional percentage sequence identity therebetween.

The present invention is also directed to a kit for identifying a sepsis-causing bacterium. The kit includes a first oligonucleotide primer pair comprising a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The first primer pair is configured to generate an amplification product that is between 45 and 200 linked nucleotides in length. The forward primer of the first primer pair is configured to hybridize with at least 70% complementarity to a first portion of a region defined by nucleotide residues 4182972 to 4183162 of Genbank gi number: 49175990 and the reverse primer configured to hybridize with at least 70% complementarity to a second portion of the region. Also included in the kit is at least one additional primer pair. The forward and reverse primers of the additional primer pair(s) are configured to hybridize to conserved sequence regions within a bacterial gene selected from the group consisting of: 16S rRNA, 23S rRNA, tufB, rpoB, valS, rplB, and gyrB.

The additional primer pair(s) of the kit may comprise at least one additional primer pairs having a forward primer and a reverse primer each between 13 to 35 linked nucleotides in length and each having at least 70% sequence identity with the corresponding forward and reverse primers of primer pair numbers 346 (SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 (SEQ ID NOs: 706:895), 349 (SEQ ID NOs: 401:1156), 360 (SEQ ID NOs: 409:1434) or 361 (SEQ ID NOs: 697:1398), 2249 (SEQ ID NOs:430:1321), 3361 (SEQ ID NOs: 1454:1468), 354 (SEQ ID NOs: 405:1072), 358 (SEQ ID NOs: 385:1093), 359 (SEQ ID NOs: 659:1250), 449 (SEQ ID NOs: 309:1336), 2249 (SEQ ID NOs: 430:1321), or 3346 (SEQ ID NOs:1448:1461).

In certain embodiments, the first oligonucleotide primer pair of the kit may comprise a forward primer and a reverse primer, each between 13 to 35 linked nucleotides in length and each having at least 70% sequence identity with the corresponding forward and reverse primers of primer pair number 3346 (SEQ ID NOs: 1448:1461); and the additional primer pair(s) may consist of at least three additional oligonucleotide primer pairs, each comprising a forward primer and a reverse primer, each between 13 to 35 linked nucleotides in length and each having at least 70% sequence identity with the corresponding forward and reverse primers of primer pair numbers, 346 (SEQ ID NOs: 202:1110), 348 (SEQ ID NOs: 560:1278), and 349 (SEQ ID NOs: 401:1156).

In certain embodiments, the kit further includes one or more additional primer pairs comprising a forward primer and a reverse primer, each between 13 to 35 linked nucleotides in length and each having at least 70% sequence identity with corresponding forward and reverse primers selected from the group consisting of primer pair numbers: 3360 (SEQ ID NOs:1444:1457), 3350 (SEQ ID NO:309:1458), 3351 (SEQ ID NOs:309:1460), 3354 (SEQ ID NO:309:1459), 3355 (SEQ ID NOs:1446:1458), 3353 (SEQ ID NOs:1447:1460), 3352 (SEQ ID NOs:1445:1458), 3347 (SEQ ID NOs:1448:1464), 3348 (SEQ ID NOs:1451:1464), 3349 (SEQ ID NOs:1450:1463), 3359 (SEQ ID NOs:1449:1462), 3358 (SEQ ID NOs:1453:1466), 3356 (SEQ ID NOs:1452:1467), 3357 (SEQ ID NOs:1452:1465), 3361 (SEQ ID NOs:1454:1468), 3362 (SEQ ID NOs:1455:1469), and 3363 (SEQ ID NOs:1456:1470).

Also disclosed is a method for identifying a sepsis-causing bacterium in a sample by amplifying a nucleic acid from the sample using an oligonucleotide primer pair that has a forward primer and a reverse primer, each between 13 and 35 linked nucleotides in length. The primer pair is configured to generate an amplification product that is between 45 and 200 linked nucleotides in length. The forward primer is configured to hybridize with at least 70% complementarity to a first portion of a region defined by nucleotide residues 4182972 to 4183162 of Genbank gi number: 49175990 and the reverse primer is configured to hybridize with at least 70% complementarity to a second portion of said region. The amplifying step generates at least one amplification product that comprises between 45 and 200 linked nucleotides. After amplification, the molecular mass of at least one amplification product is determined by mass spectrometry.

In some embodiments, the method further includes comparing the molecular mass to a database comprising a plurality of molecular masses of bioagent identifying amplicons. A match between the determined molecular mass and a molecular mass included in the database identifies the sepsis-causing bacterium in the sample.

In some embodiments, the method further includes calculating a base composition of the amplification product using the determined molecular mass. The base composition may then be compared with calculated base compositions. A match between a calculated base composition and a base composition included in the database identifies the sepsis-causing bacterium in the sample.

In some embodiments, the method uses a forward primer that has at least 70% sequence identity with SEQ ID NO: 1448.

In some embodiments, the method uses a reverse primer that has at least 70% sequence identity with SEQ ID NO: 1461.

In some embodiments, the method further includes repeating the amplifying and determining steps using at least one additional oligonucleotide primer pair. The forward and reverse primers of the additional primer pair are designed to hybridize to conserved sequence regions within a bacterial gene selected from the group consisting of 16S rRNA, 23S rRNA, tufB rpoB, valS, rplB, and gyrB.

In some embodiments of the method, the molecular mass identifies the presence of said sepsis-causing bacterium in said sample.

In some embodiments, the method further comprises determining either the sensitivity or the resistance of the sepsis-causing bacterium to one or more antibiotics.

In some embodiments, the method of claim 35, wherein said molecular mass identifies a sub-species characteristic, strain, or genotype of said sepsis-causing bacterium in said sample.

Also disclosed herein is a method for identification of a sepsis-causing bacterium in a sample by obtaining a plurality of amplification products using one or more primer pairs that hybridize to ribosomal RNA and one or more primer pairs that hybridize to a housekeeping gene. The molecular masses of the plurality of amplification products are measured and base compositions of the amplification products are calculated from the molecular masses. Comparison of the base compositions to known base compositions of amplification products of known sepsis-causing bacteria produced with the primer pairs thereby identifies the sepsis-causing bacterium in the sample.

In some embodiments, the molecular masses are measured by mass spectrometry such as electrospray time-of-flight mass spectrometry for example.

In some embodiments, the housekeeping genes include rpoC, valS, rpoB, rplB, gyrA or tufB.

In some embodiments, the primers of the primer pairs that hybridize to ribosomal RNA are 13 to 35 nucleobases in length and have at least 70% sequence identity with the corresponding member of primer pair number 346 (SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 (SEQ ID NOs: 706:895), 349 (SEQ ID NOs: 401:1156), 360 (SEQ ID NOs: 409:1434) or 361 (SEQ ID NOs: 697:1398).

In some embodiments, the primers of the primer pairs that hybridize to a housekeeping gene are between 13 to 35 nucleobases in length and have at least 70% sequence identity with the corresponding member of primer pair number 354 (SEQ ID NOs: 405:1072), 358 (SEQ ID NOs: 385:1093), 359 (SEQ ID NOs: 659:1250), 449 (SEQ ID NOs: 309:1336) or 2249 (SEQ ID NOs: 430:1321).

In some embodiments of the method, the sepsis-causing bacterium is Bacteroides fragilis, Prevotella denticola, Porphyromonas gingivalis, Borrelia burgdorferi, Mycobacterium tuburculosis, Mycobacterium fortuitum, Corynebacteriumjeikeium, Propionibacterium acnes, Mycoplasma pneumoniae, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus mitis, Streptococcus pyogenes, Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus coagulase-negative, Staphylococcus epidermis, Staphylococcus hemolyticus, Campylobacter jejuni, Bordatella pertussis, Burkholderia cepacia, Legionella pneumophila, Acinetobacter baumannii, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, Aeromonas hydrophila, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Moxarella catarrhalis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Pantoea agglomerans, Bartonella henselae, Stenotrophomonas maltophila, Actinobacillus actinomycetemcomitans, Haemophilus influenzae, Escherichia coli, Klebsiella oxytoca, Serratia marcescens or Yersinia enterocolitica.

Also disclosed is a kit for identification of a sepsis-causing bacterium. The kit includes one or more primer pairs that hybridize to ribosomal RNA. Each member of the primer pairs is between 13 to 35 nucleobases in length and has at least 70% sequence identity with the corresponding member of primer pair number 346 (SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 (SEQ ID NOs: 706:895), 349 (SEQ ID NOs: 401:1156), 360 (SEQ ID NOs: 409:1434) or 361 (SEQ ID NOs: 697:1398).

The kit may also include one or more additional primer pairs that hybridize to housekeeping genes. The forward and reverse primers of the additional primer pairs are between 13 to 35 nucleobases in length and have at least 70% sequence identity with the corresponding member of primer pair number 354 (SEQ ID NOs: 405:1072), 358 (SEQ ID NOs: 385:1093), 359 (SEQ ID NOs: 659:1250), 449 (SEQ ID NOs: 309:1336), 2249 (SEQ ID NOs: 430:1321), 3346 (SEQ ID NOs:1448:1461), or 3361 (SEQ ID NOs: 1454:1468).

Some embodiments are methods for determination of the quantity of an unknown bacterium in a sample. The sample is contacted with the composition described above and a known quantity of a calibration polynucleotide comprising a calibration sequence. Nucleic acid from the unknown bacterium in the sample is concurrently amplified with the composition described above and nucleic acid from the calibration polynucleotide in the sample is concurrently amplified with the composition described above to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon. The molecular masses and abundances for the bacterial bioagent identifying amplicon and the calibration amplicon are determined. The bacterial bioagent identifying amplicon is distinguished from the calibration amplicon based on molecular mass and comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in the sample. In some embodiments, the base composition of the bacterial bioagent identifying amplicon is determined.

Some embodiments are methods for detecting or quantifying bacteria by combining a nucleic acid amplification process with a mass determination process. In some embodiments, such methods identify or otherwise analyze the bacterium by comparing mass information from an amplification product with a calibration or control product. Such methods can be carried out in a highly multiplexed and/or parallel manner allowing for the analysis of as many as 300 samples per 24 hours on a single mass measurement platform. The accuracy of the mass determination methods permits allows for the ability to discriminate between different bacteria such as, for example, various genotypes and drug resistant strains of sepsis-causing bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description, is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation.

FIG. 1: process diagram illustrating a representative primer pair selection process.

FIG. 2: process diagram illustrating an embodiment of the calibration method.

FIG. 3: common pathogenic bacteria and primer pair coverage. The primer pair number in the upper right hand corner of each polygon indicates that the primer pair can produce a bioagent identifying amplicon for all species within that polygon.

FIG. 4: a representative 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples (labeled NHRC samples) closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.

FIG. 5: a representative mass spectrum of amplification products indicating the presence of bioagent identifying amplicons of Streptococcus pyogenes, Neisseria meningitidis, and Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses and base compositions for the sense strand of each amplification product are shown.

FIG. 6: a representative mass spectrum of amplification products representing a bioagent identifying amplicon of Streptococcus pyogenes, and a calibration amplicon obtained from amplification of nucleic acid from a clinical sample with primer pair number 356 which targets rplB. The experimentally determined molecular mass and base composition for the sense strand of the Streptococcus pyogenes amplification product is shown.

FIG. 7: a representative mass spectrum of an amplified nucleic acid mixture which contained the Ames strain of Bacillus anthracis, a known quantity of combination calibration polynucleotide (SEQ ID NO: 1464), and primer pair number 350 which targets the capC gene on the virulence plasmid pX02 of Bacillus anthracis. Calibration amplicons produced in the amplification reaction are visible in the mass spectrum as indicated and abundance data (peak height) are used to calculate the quantity of the Ames strain of Bacillus anthracis.

DEFINITIONS

As used herein, the term “abundance” refers to an amount. The amount may be described in terms of concentration which are common in molecular biology such as “copy number,” “pfu or plate-forming unit” which are well known to those with ordinary skill. Concentration may be relative to a known standard or may be absolute.

As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” also comprises “sample template.”

As used herein the term “amplification” refers to a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Qβ replicase, MDV-1 RNA is the specific template for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (D. Y. Wu and R. B. Wallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]).

As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification, excluding primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

As used herein, the term “analogous” when used in context of comparison of bioagent identifying amplicons indicates that the bioagent identifying amplicons being compared are produced with the same pair of primers. For example, bioagent identifying amplicon “A” and bioagent identifying amplicon “B”, produced with the same pair of primers are analogous with respect to each other. Bioagent identifying amplicon “C”, produced with a different pair of primers is not analogous to either bioagent identifying amplicon “A” or bioagent identifying amplicon “B”.

As used herein, the term “anion exchange functional group” refers to a positively charged functional group capable of binding an anion through an electrostatic interaction. The most well known anion exchange functional groups are the amines, including primary, secondary, tertiary and quaternary amines.

The term “bacteria” or “bacterium” refers to any member of the groups of eubacteria and archaebacteria.

As used herein, a “base composition” is the exact number of each nucleobase (for example, A, T, C and G) in a segment of nucleic acid. For example, amplification of nucleic acid of Staphylococcus aureus strain carrying the lukS-PV gene with primer pair number 2095 (SEQ ID NOs: 456:1261) produces an amplification product 117 nucleobases in length from nucleic acid of the lukS-PV gene that has a base composition of A35 G17 C19 T46 (by convention—with reference to the sense strand of the amplification product). Because the molecular masses of each of the four natural nucleotides and chemical modifications thereof are known (if applicable), a measured molecular mass can be deconvoluted to a list of possible base compositions. Identification of a base composition of a sense strand which is complementary to the corresponding antisense strand in terms of base composition provides a confirmation of the true base composition of an unknown amplification product. For example, the base composition of the antisense strand of the 139 nucleobase amplification product described above is A46 G19 C17 T35.

As used herein, a “base composition probability cloud” is a representation of the diversity in base composition resulting from a variation in sequence that occurs among different isolates of a given species. The “base composition probability cloud” represents the base composition constraints for each species and is typically visualized using a pseudo four-dimensional plot.

As used herein, a “bioagent” is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus. Examples of bioagents include, but are not limited, to cells, (including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered. As used herein, a “pathogen” is a bioagent which causes a disease or disorder.

As used herein, a “bioagent division” is defined as group of bioagents above the species level and includes but is not limited to, orders, families, classes, clades, genera or other such groupings of bioagents above the species level.

As used herein, the term “bioagent identifying amplicon” refers to a polynucleotide that is amplified from a bioagent in an amplification reaction and which 1) provides sufficient variability to distinguish among bioagents from whose nucleic acid the bioagent identifying amplicon is produced and 2) whose molecular mass is amenable to a rapid and convenient molecular mass determination modality such as mass spectrometry, for example.

As used herein, the term “biological product” refers to any product originating from an organism. Biological products are often products of processes of biotechnology. Examples of biological products include, but are not limited to: cultured cell lines, cellular components, antibodies, proteins and other cell-derived biomolecules, growth media, growth harvest fluids, natural products and bio-pharmaceutical products.

The terms “biowarfare agent” and “bioweapon” are synonymous and refer to a bacterium, virus, fungus or protozoan that could be deployed as a weapon to cause bodily harm to individuals. Military or terrorist groups may be implicated in deployment of biowarfare agents.

As used herein, the term “broad range survey primer pair” refers to a primer pair designed to produce bioagent identifying amplicons across different broad groupings of bioagents. For example, the ribosomal RNA-targeted primer pairs are broad range survey primer pairs which have the capability of producing bacterial bioagent identifying amplicons for essentially all known bacteria. With respect to broad range primer pairs employed for identification of bacteria, a broad range survey primer pair for bacteria such as 16S rRNA primer pair number 346 (SEQ ID NOs: 202:1110) for example, will produce an bacterial bioagent identifying amplicon for essentially all known bacteria.

The term “calibration amplicon” refers to a nucleic acid segment representing an amplification product obtained by amplification of a calibration sequence with a pair of primers designed to produce a bioagent identifying amplicon.

The term “calibration sequence” refers to a polynucleotide sequence to which a given pair of primers hybridizes for the purpose of producing an internal (i.e.: included in the reaction) calibration standard amplification product for use in determining the quantity of a bioagent in a sample. The calibration sequence may be expressly added to an amplification reaction, or may already be present in the sample prior to analysis.

The term “clade primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for species belonging to a clade group. A clade primer pair may also be considered as a “speciating” primer pair which is useful for distinguishing among closely related species.

The term “codon” refers to a set of three adjoined nucleotides (triplet) that codes for an amino acid or a termination signal.

As used herein, the term “codon base composition analysis,” refers to determination of the base composition of an individual codon by obtaining a bioagent identifying amplicon that includes the codon. The bioagent identifying amplicon will at least include regions of the target nucleic acid sequence to which the primers hybridize for generation of the bioagent identifying amplicon as well as the codon being analyzed, located between the two primer hybridization regions.

As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence “5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.

The term “complement of a nucleic acid sequence” as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5′ end of one sequence is paired with the 3′ end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids disclosed herein and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. Where a first oligonucleotide is complementary to a region of a target nucleic acid and a second oligonucleotide has complementary to the same region (or a portion of this region) a “region of overlap” exists along the target nucleic acid. The degree of overlap will vary depending upon the extent of the complementarity.

As used herein, the term “division-wide primer pair” refers to a primer pair designed to produce bioagent identifying amplicons within sections of a broader spectrum of bioagents For example, primer pair number 352 (SEQ ID NOs: 687:1411), a division-wide primer pair, is designed to produce bacterial bioagent identifying amplicons for members of the Bacillus group of bacteria which comprises, for example, members of the genera Streptococci, Enterococci, and Staphylococci. Other division-wide primer pairs may be used to produce bacterial bioagent identifying amplicons for other groups of bacterial bioagents.

As used herein, the term “concurrently amplifying” used with respect to more than one amplification reaction refers to the act of simultaneously amplifying more than one nucleic acid in a single reaction mixture.

As used herein, the term “drill-down primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for identification of sub-species characteristics or conformation of a species assignment. For example, primer pair number 2146 (SEQ ID NOs: 437:1137), a drill-down Staphylococcus aureus genotyping primer pair, is designed to produce Staphylococcus aureus genotyping amplicons. Other drill-down primer pairs may be used to produce bioagent identifying amplicons for Staphylococcus aureus and other bacterial species.

The term “duplex” refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding the their complementary bases arrayed on a second strand. The condition of being in a duplex form reflects on the state of the bases of a nucleic acid. By virtue of base pairing, the strands of nucleic acid also generally assume the tertiary structure of a double helix, having a major and a minor groove. The assumption of the helical form is implicit in the act of becoming duplexed.

As used herein, the term “etiology” refers to the causes or origins, of diseases or abnormal physiological conditions.

The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide or a precursor. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.

The terms “homology,” “homologous” and “sequence identity” refer to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is otherwise identical to another 20 nucleobase primer but having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of a primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer. As used herein, sequence identity is meant to be properly determined when the query sequence and the subject sequence are both described and aligned in the 5′ to 3′ direction. Sequence alignment algorithms such as BLAST, will return results in two different alignment orientations. In the Plus/Plus orientation, both the query sequence and the subject sequence are aligned in the 5′ to 3′ direction. On the other hand, in the Plus/Minus orientation, the query sequence is in the 5′ to 3′ direction while the subject sequence is in the 3′ to 5′ direction. It should be understood that with respect to the primers disclosed herein, sequence identity is properly determined when the alignment is designated as Plus/Plus. Sequence identity may also encompass alternate or modified nucleobases that perform in a functionally similar manner to the regular nucleobases adenine, thymine, guanine and cytosine with respect to hybridization and primer extension in amplification reactions. In a non-limiting example, if the 5-propynyl pyrimidines propyne C and/or propyne T replace one or more C or T residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. In another non-limiting example, Inosine (I) may be used as a replacement for G or T and effectively hybridize to C, A or U (uracil). Thus, if inosine replaces one or more C, A or U residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. Other such modified or universal bases may exist which would perform in a functionally similar manner for hybridization and amplification reactions and will be understood to fall within this definition of sequence identity.

As used herein, “housekeeping gene” refers to a gene encoding a protein or RNA involved in basic functions required for survival and reproduction of a bioagent. Housekeeping genes include, but are not limited to genes encoding RNA or proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like.

As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the T_(m) of the formed hybrid. “Hybridization” methods involve the annealing of one nucleic acid to another, complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.

The term “in silico” refers to processes taking place via computer calculations. For example, electronic PCR (ePCR) is a process analogous to ordinary PCR except that it is carried out using nucleic acid sequences and primer pair sequences stored on a computer formatted medium.

As used herein, “intelligent primers” are primers that are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and, upon amplification, yield amplification products which ideally provide enough variability to distinguish individual bioagents, and which are amenable to molecular mass analysis. By the term “highly conserved,” it is meant that the sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity among all, or at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of species or strains.

The “ligase chain reaction” (LCR; sometimes referred to as “Ligase Amplification Reaction” (LAR) described by Barany, Proc. Natl. Acad. Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wu and Wallace, Genomics 4:560 (1989) has developed into a well-recognized alternative method for amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, that hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, hybridization and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes. However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.

The term “locked nucleic acid” or “LNA” refers to a nucleic acid analogue containing one or more 2′-O, 4′-C-methylene-β-D-ribofuranosyl nucleotide monomers in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The primers disclosed herein may contain LNA modifications.

As used herein, the term “mass-modifying tag” refers to any modification to a given nucleotide which results in an increase in mass relative to the analogous non-mass modified nucleotide. Mass-modifying tags can include heavy isotopes of one or more elements included in the nucleotide such as carbon-13 for example. Other possible modifications include addition of substituents such as iodine or bromine at the 5 position of the nucleobase for example.

The term “mass spectrometry” refers to measurement of the mass of atoms or molecules. The molecules are first converted to ions, which are separated using electric or magnetic fields according to the ratio of their mass to electric charge. The measured masses are used to identity the molecules.

The term “microorganism” as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, virus, protozoans, fungi; and ciliates.

The term “multi-drug resistant” or multiple-drug resistant” refers to a microorganism which is resistant to more than one of the antibiotics or antimicrobial agents used in the treatment of said microorganism.

The term “multiplex PCR” refers to a PCR reaction where more than one primer set is included in the reaction pool allowing 2 or more different DNA targets to be amplified by PCR in a single reaction tube.

The term “non-template tag” refers to a stretch of at least three guanine or cytosine nucleobases of a primer used to produce a bioagent identifying amplicon which are not complementary to the template. A non-template tag is incorporated into a primer for the purpose of increasing the primer-duplex stability of later cycles of amplification by incorporation of extra G-C pairs which each have one additional hydrogen bond relative to an A-T pair.

The term “nucleic acid sequence” as used herein refers to the linear composition of the nucleic acid residues A, T, C or G or any modifications thereof, within an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or antisense strand

As used herein, the term “nucleobase” is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).

The term “nucleotide analog” as used herein refers to modified or non-naturally occurring nucleotides such as 5-propynyl pyrimidines (i.e., 5-propynyl-dTTP and 5-propynyl-dTCP), 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides.

The term “oligonucleotide” as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 13 to 35 nucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5′-end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′-end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3′ end of the first region is before the 5′ end of the second region when moving along a strand of nucleic acid in a 5′ to 3′ direction. All oligonucleotide primers disclosed herein are understood to be presented in the 5′ to 3′ direction when reading left to right. When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3′ end of one oligonucleotide points towards the 5′ end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide. Similarly, when two overlapping oligonucleotides are hybridized to the same linear complementary nucleic acid sequence, with the first oligonucleotide positioned such that its 5′ end is upstream of the 5′ end of the second oligonucleotide, and the 3′ end of the first oligonucleotide is upstream of the 3′ end of the second oligonucleotide, the first oligonucleotide may be called the “upstream” oligonucleotide and the second oligonucleotide may be called the “downstream” oligonucleotide.

As used herein, a “pathogen” is a bioagent which causes a disease or disorder.

As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

The term “peptide nucleic acid” (“PNA”) as used herein refers to a molecule comprising bases or base analogs such as would be found in natural nucleic acid, but attached to a peptide backbone rather than the sugar-phosphate backbone typical of nucleic acids. The attachment of the bases to the peptide is such as to allow the bases to base pair with complementary bases of nucleic acid in a manner similar to that of an oligonucleotide. These small molecules, also designated anti gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63). The primers disclosed herein may comprise PNAs.

The term “polymerase” refers to an enzyme having the ability to synthesize a complementary strand of nucleic acid from a starting template nucleic acid strand and free dNTPs.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.” With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

The term “polymerization means” or “polymerization agent” refers to any agent capable of facilitating the addition of nucleoside triphosphates to an oligonucleotide. Preferred polymerization means comprise DNA and RNA polymerases.

As used herein, the terms “pair of primers,” or “primer pair” are synonymous. A primer pair is used for amplification of a nucleic acid sequence. A pair of primers comprises a forward primer and a reverse primer. The forward primer hybridizes to a sense strand of a target gene sequence to be amplified and primes synthesis of an antisense strand (complementary to the sense strand) using the target sequence as a template. A reverse primer hybridizes to the antisense strand of a target gene sequence to be amplified and primes synthesis of a sense strand (complementary to the antisense strand) using the target sequence as a template.

The primers are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which ideally provide enough variability to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus design of the primers requires selection of a variable region with appropriate variability to resolve the identity of a given bioagent. Bioagent identifying amplicons are ideally specific to the identity of the bioagent.

Properties of the primers may include any number of properties related to structure including, but not limited to: nucleobase length which may be contiguous (linked together) or non-contiguous (for example, two or more contiguous segments which are joined by a linker or loop moiety), modified or universal nucleobases (used for specific purposes such as for example, increasing hybridization affinity, preventing non-templated adenylation and modifying molecular mass) percent complementarity to a given target sequences.

Properties of the primers also include functional features including, but not limited to, orientation of hybridization (forward or reverse) relative to a nucleic acid template. The coding or sense strand is the strand to which the forward priming primer hybridizes (forward priming orientation) while the reverse priming primer hybridizes to the non-coding or antisense strand (reverse priming orientation). The functional properties of a given primer pair also include the generic template nucleic acid to which the primer pair hybridizes. For example, identification of bioagents can be accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Other primers may have the functionality of producing bioagent identifying amplicons for members of a given taxonomic genus, clade, species, sub-species or genotype (including genetic variants which may include presence of virulence genes or antibiotic resistance genes or mutations). Additional functional properties of primer pairs include the functionality of performing amplification either singly (single primer pair per amplification reaction vessel) or in a multiplex fashion (multiple primer pairs and multiple amplification reactions within a single reaction vessel).

As used herein, the terms “purified” or “substantially purified” refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” or “isolated oligonucleotide” is therefore a substantially purified polynucleotide.

The term “reverse transcriptase” refers to an enzyme having the ability to transcribe DNA from an RNA template. This enzymatic activity is known as reverse transcriptase activity. Reverse transcriptase activity is desirable in order to obtain DNA from RNA viruses which can then be amplified and analyzed by the methods disclosed herein.

The term “ribosomal RNA” or “rRNA” refers to the primary ribonucleic acid constituent of ribosomes. Ribosomes are the protein-manufacturing organelles of cells and exist in the cytoplasm. Ribosomal RNAs are transcribed from the DNA genes encoding them.

The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc. Environmental samples include environmental material such as surface matter, soil, water, air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the methods disclosed herein. The term “source of target nucleic acid” refers to any sample that contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.

As used herein, the term “sample template” refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is often a contaminant. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

A “segment” is defined herein as a region of nucleic acid within a target sequence.

The “self-sustained sequence replication reaction” (3SR) (Guatelli et al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based in vitro amplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177 [1989]) that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5′ end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).

As used herein, the term ““sequence alignment”” refers to a listing of multiple DNA or amino acid sequences and aligns them to highlight their similarities. The listings can be made using bioinformatics computer programs.

As used herein, the terms “sepsis” and “septicemia refer to disease caused by the spread of bacteria and their toxins in the bloodstream. For example, a “sepsis-causing bacterium” is the causative agent of sepsis i.e. the bacterium infecting the bloodstream of an individual with sepsis.

As used herein, the term “speciating primer pair” refers to a primer pair designed to produce a bioagent identifying amplicon with the diagnostic capability of identifying species members of a group of genera or a particular genus of bioagents. Primer pair number 2249 (SEQ ID NOs: 430:1321), for example, is a speciating primer pair used to distinguish Staphylococcus aureus from other species of the genus Staphylococcus.

As used herein, a “sub-species characteristic” is a genetic characteristic that provides the means to distinguish two members of the same bioagent species. For example, one viral strain could be distinguished from another viral strain of the same species by possessing a genetic change (e.g., for example, a nucleotide deletion, addition or substitution) in one of the viral genes, such as the RNA-dependent RNA polymerase. Sub-species characteristics such as virulence genes and drug—are responsible for the phenotypic differences among the different strains of bacteria.

As used herein, the term “target” is used in a broad sense to indicate the gene or genomic region being amplified by the primers. Because the methods disclosed herein provide a plurality of amplification products from any given primer pair (depending on the bioagent being analyzed), multiple amplification products from different specific nucleic acid sequences may be obtained. Thus, the term “target” is not used to refer to a single specific nucleic acid sequence. The “target” is sought to be sorted out from other nucleic acid sequences and contains a sequence that has at least partial complementarity with an oligonucleotide primer. The target nucleic acid may comprise single- or double-stranded DNA or RNA. A “segment” is defined as a region of nucleic acid within the target sequence.

The term “template” refers to a strand of nucleic acid on which a complementary copy is built from nucleoside triphosphates through the activity of a template-dependent nucleic acid polymerase. Within a duplex the template strand is, by convention, depicted and described as the “bottom” strand. Similarly, the non-template strand is often depicted and described as the “top” strand.

As used herein, the term “T_(m)” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. Several equations for calculating the T_(m) of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the T_(m) value may be calculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr. Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36, 10581-94 (1997) include more sophisticated computations which take structural and environmental, as well as sequence characteristics into account for the calculation of T_(m).

The term “triangulation genotyping analysis” refers to a method of genotyping a bioagent by measurement of molecular masses or base compositions of amplification products, corresponding to bioagent identifying amplicons, obtained by amplification of regions of more than one gene. In this sense, the term “triangulation” refers to a method of establishing the accuracy of information by comparing three or more types of independent points of view bearing on the same findings. Triangulation genotyping analysis carried out with a plurality of triangulation genotyping analysis primers yields a plurality of base compositions that then provide a pattern or “barcode” from which a species type can be assigned. The species type may represent a previously known sub-species or strain, or may be a previously unknown strain having a specific and previously unobserved base composition barcode indicating the existence of a previously unknown genotype.

As used herein, the term “triangulation genotyping analysis primer pair” is a primer pair designed to produce bioagent identifying amplicons for determining species types in a triangulation genotyping analysis.

The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by analyzing a plurality of bioagent identifying amplicons produced with different primer pairs. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.

As used herein, the term “unknown bioagent” may mean either: (i) a bioagent whose existence is known (such as the well known bacterial species Staphylococcus aureus for example) but which is not known to be in a sample to be analyzed, or (ii) a bioagent whose existence is not known (for example, the SARS coronavirus was unknown prior to April 2003). For example, if the method for identification of coronaviruses disclosed in commonly owned U.S. patent Ser. No. 10/829,826 (incorporated herein by reference in its entirety) was to be employed prior to April 2003 to identify the SARS coronavirus in a clinical sample, both meanings of “unknown” bioagent are applicable since the SARS coronavirus was unknown to science prior to April, 2003 and since it was not known what bioagent (in this case a coronavirus) was present in the sample. On the other hand, if the method of U.S. patent Ser. No. 10/829,826 was to be employed subsequent to April 2003 to identify the SARS coronavirus in a clinical sample, only the first meaning (i) of “unknown” bioagent would apply since the SARS coronavirus became known to science subsequent to April 2003 and since it was not known what bioagent was present in the sample.

The term “variable sequence” as used herein refers to differences in nucleic acid sequence between two nucleic acids. For example, the genes of two different bacterial species may vary in sequence by the presence of single base substitutions and/or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. As used herein, the term “viral nucleic acid” includes, but is not limited to, DNA, RNA, or DNA that has been obtained from viral RNA, such as, for example, by performing a reverse transcription reaction. Viral RNA can either be single-stranded (of positive or negative polarity) or double-stranded.

The term “virus” refers to obligate, ultramicroscopic, parasites that are incapable of autonomous replication (i.e., replication requires the use of the host cell's machinery). Viruses can survive outside of a host cell but cannot replicate.

The term “wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified”, “mutant” or “polymorphic” refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

As used herein, a “wobble base” is a variation in a codon found at the third nucleotide position of a DNA triplet. Variations in conserved regions of sequence are often found at the third nucleotide position due to redundancy in the amino acid code.

DETAILED DESCRIPTION OF EMBODIMENTS

A. Bioagent Identifying Amplicons

Disclosed herein are methods for detection and identification of unknown bioagents using bioagent identifying amplicons. Primers are selected to hybridize to conserved sequence regions of nucleic acids derived from a bioagent, and which bracket variable sequence regions to yield a bioagent identifying amplicon, which can be amplified and which is amenable to molecular mass determination. The molecular mass then provides a means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent. The molecular mass or corresponding base composition signature of the amplification product is then matched against a database of molecular masses or base composition signatures. A match is obtained when an experimentally-determined molecular mass or base composition of an analyzed amplification product is compared with known molecular masses or base compositions of known bioagent identifying amplicons and the experimentally determined molecular mass or base composition is the same as the molecular mass or base composition of one of the known bioagent identifying amplicons. Alternatively, the experimentally-determined molecular mass or base composition may be within experimental error of the molecular mass or base composition of a known bioagent identifying amplicon and still be classified as a match. In some cases, the match may also be classified using a probability of match model such as the models described in U.S. Ser. No. 11/073,362, which is commonly owned and incorporated herein by reference in entirety. Furthermore, the method can be applied to rapid parallel multiplex analyses, the results of which can be employed in a triangulation identification strategy. The present method provides rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent detection and identification.

Despite enormous biological diversity, all forms of life on earth share sets of essential, common features in their genomes. Since genetic data provide the underlying basis for identification of bioagents by the methods disclosed herein, it is necessary to select segments of nucleic acids which ideally provide enough variability to distinguish each individual bioagent and whose molecular mass is amenable to molecular mass determination.

Unlike bacterial genomes, which exhibit conservation of numerous genes (i.e. housekeeping genes) across all organisms, viruses do not share a gene that is essential and conserved among all virus families. Therefore, viral identification is achieved within smaller groups of related viruses, such as members of a particular virus family or genus. For example, RNA-dependent RNA polymerase is present in all single-stranded RNA viruses and can be used for broad priming as well as resolution within the virus family.

In some embodiments, at least one bacterial nucleic acid segment is amplified in the process of identifying the bacterial bioagent. Thus, the nucleic acid segments that can be amplified by the primers disclosed herein and that provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as bioagent identifying amplicons.

In some embodiments, bioagent identifying amplicons comprise from about 45 to about 200 nucleobases (i.e. from about 45 to about 200 linked nucleosides), although both longer and short regions may be used. One of ordinary skill in the art will appreciate that these embodiments include compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 nucleobases in length, or any range therewithin.

It is the combination of the portions of the bioagent nucleic acid segment to which the primers hybridize (hybridization sites) and the variable region between the primer hybridization sites that comprises the bioagent identifying amplicon. Thus, it can be said that a given bioagent identifying amplicon is “defined by” a given pair of primers.

In some embodiments, bioagent identifying amplicons amenable to molecular mass determination which are produced by the primers described herein are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to obtain predictable fragments of a length compatible with the particular mode of molecular mass determination. Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with chemical reagents, restriction enzymes or cleavage primers, for example. Thus, in some embodiments, bioagent identifying amplicons are larger than 200 nucleobases and are amenable to molecular mass determination following restriction digestion. Methods of using restriction enzymes and cleavage primers are well known to those with ordinary skill in the art.

In some embodiments, amplification products corresponding to bioagent identifying amplicons are obtained using the polymerase chain reaction (PCR) that is a routine method to those with ordinary skill in the molecular biology arts. Other amplification methods may be used such as ligase chain reaction (LCR), low-stringency single primer PCR, and multiple strand displacement amplification (MDA). These methods are also known to those with ordinary skill.

B. Primers and Primer Pairs

In some embodiments, the primers are designed to bind to conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which provide variability sufficient to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus, design of the primers involves selection of a variable region with sufficient variability to resolve the identity of a given bioagent. In some embodiments, bioagent identifying amplicons are specific to the identity of the bioagent.

In some embodiments, identification of bioagents is accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Examples of broad range survey primers include, but are not limited to: primer pair numbers: 346 (SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 SEQ ID NOs: 706:895), and 361 (SEQ ID NOs: 697:1398) which target DNA encoding 16S rRNA, and primer pair numbers 349 (SEQ ID NOs: 401:1156) and 360 (SEQ ID NOs: 409:1434) which target DNA encoding 23S rRNA.

In some embodiments, drill-down primers are designed with the objective of identifying a bioagent at the sub-species level (including strains, subtypes, variants and isolates) based on sub-species characteristics which may, for example, include single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs), deletions, drug resistance mutations or any other modification of a nucleic acid sequence of a bioagent relative to other members of a species having different sub-species characteristics. Drill-down intelligent primers are not always required for identification at the sub-species level because broad range survey intelligent primers may, in some cases provide sufficient identification resolution to accomplishing this identification objective. Examples of drill-down primers include, but are not limited to: confirmation primer pairs such as primer pair numbers 351 (SEQ ID NOs: 355:1423) and 353 (SEQ ID NOs: 220:1394), which target the pX01 virulence plasmid of Bacillus anthracis. Other examples of drill-down primer pairs are found in sets of triangulation genotyping primer pairs such as, for example, the primer pair number 2146 (SEQ ID NOs: 437:1137) which targets the arcC gene (encoding carmabate kinase) and is included in an 8 primer pair panel or kit for use in genotyping Staphylococcus aureus, or in other panels or kits of primer pairs used for determining drug-resistant bacterial strains, such as, for example, primer pair number 2095 (SEQ ID NOs: 456:1261) which targets the pv-luk gene (encoding Panton-Valentine leukocidin) and is included in an 8 primer pair panel or kit for use in identification of drug resistant strains of Staphylococcus aureus.

A representative process flow diagram used for primer selection and validation process is outlined in FIG. 1. For each group of organisms, candidate target sequences are identified (200) from which nucleotide alignments are created (210) and analyzed (220). Primers are then designed by selecting appropriate priming regions (230) to facilitate the selection of candidate primer pairs (240). The primer pairs are then subjected to in silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are obtained from sequence databases such as GenBank or other sequence collections (310) and checked for specificity in silico (320). Bioagent identifying amplicons obtained from GenBank sequences (310) can also be analyzed by a probability model which predicts the capability of a given amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are then stored in a base composition database (325). Alternatively, base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database (330). Candidate primer pairs (240) are validated by testing their ability to hybridize to target nucleic acid by an in vitro amplification by a method such as PCR analysis (400) of nucleic acid from a collection of organisms (410). Amplification products thus obtained are analyzed by gel electrophoresis or by mass spectrometry to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products (420).

Many of the important pathogens, including the organisms of greatest concern as biowarfare agents, have been completely sequenced. This effort has greatly facilitated the design of primers for the detection of unknown bioagents. The combination of broad-range priming with division-wide and drill-down priming has been used very successfully in several applications of the technology, including environmental surveillance for biowarfare threat agents and clinical sample analysis for medically important pathogens.

Synthesis of primers is well known and routine in the art. The primers may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.

In some embodiments, primers are employed as compositions for use in methods for identification of bacterial bioagents as follows: a primer pair composition is contacted with nucleic acid (such as, for example, bacterial DNA or DNA reverse transcribed from the rRNA) of an unknown bacterial bioagent. The nucleic acid is then amplified by a nucleic acid amplification technique, such as PCR for example, to obtain an amplification product that represents a bioagent identifying amplicon. The molecular mass of each strand of the double-stranded amplification product is determined by a molecular mass measurement technique such as mass spectrometry for example, wherein the two strands of the double-stranded amplification product are separated during the ionization process. In some embodiments, the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray time of flight mass spectrometry (ESI-TOF-MS). A list of possible base compositions can be generated for the molecular mass value obtained for each strand and the choice of the correct base composition from the list is facilitated by matching the base composition of one strand with a complementary base composition of the other strand. The molecular mass or base composition thus determined is then compared with a database of molecular masses or base compositions of analogous bioagent identifying amplicons for known viral bioagents. A match between the molecular mass or base composition of the amplification product and the molecular mass or base composition of an analogous bioagent identifying amplicon for a known viral bioagent indicates the identity of the unknown bioagent. In some embodiments, the primer pair used is one of the primer pairs of Table 2. In some embodiments, the method is repeated using one or more different primer pairs to resolve possible ambiguities in the identification process or to improve the confidence level for the identification assignment.

In some embodiments, a bioagent identifying amplicon may be produced using only a single primer (either the forward or reverse primer of any given primer pair), provided an appropriate amplification method is chosen, such as, for example, low stringency single primer PCR (LSSP-PCR). Adaptation of this amplification method in order to produce bioagent identifying amplicons can be accomplished by one with ordinary skill in the art without undue experimentation.

In some embodiments, the oligonucleotide primers are broad range survey primers which hybridize to conserved regions of nucleic acid encoding the hexon gene of all (or between 80% and 100%, between 85% and 100%, between 90% and 100% or between 95% and 100%) known bacteria and produce bacterial bioagent identifying amplicons.

In some cases, the molecular mass or base composition of a bacterial bioagent identifying amplicon defined by a broad range survey primer pair does not provide enough resolution to unambiguously identify a bacterial bioagent at or below the species level. These cases benefit from further analysis of one or more bacterial bioagent identifying amplicons generated from at least one additional broad range survey primer pair or from at least one additional division-wide primer pair. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as triangulation identification.

In other embodiments, the oligonucleotide primers are division-wide primers which hybridize to nucleic acid encoding genes of species within a genus of bacteria. In other embodiments, the oligonucleotide primers are drill-down primers which enable the identification of sub-species characteristics. Drill down primers provide the functionality of producing bioagent identifying amplicons for drill-down analyses such as strain typing when contacted with nucleic acid under amplification conditions. Identification of such sub-species characteristics is often critical for determining proper clinical treatment of viral infections. In some embodiments, sub-species characteristics are identified using only broad range survey primers and division-wide and drill-down primers are not used.

In some embodiments, the primers used for amplification hybridize to and amplify genomic DNA, and DNA of bacterial plasmids.

In some embodiments, various computer software programs may be used to aid in design of primers for amplification reactions such as Primer Premier 5 (Premier Biosoft, Palo Alto, Calif.) or OLIGO Primer Analysis Software (Molecular Biology Insights, Cascade, Colo.). These programs allow the user to input desired hybridization conditions such as melting temperature of a primer-template duplex for example. In some embodiments, an in silico PCR search algorithm, such as (ePCR) is used to analyze primer specificity across a plurality of template sequences which can be readily obtained from public sequence databases such as GenBank for example. An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs. In some embodiments, the hybridization conditions applied to the algorithm can limit the results of primer specificity obtained from the algorithm. In some embodiments, the melting temperature threshold for the primer template duplex is specified to be 35° C. or a higher temperature. In some embodiments the number of acceptable mismatches is specified to be seven mismatches or less. In some embodiments, the buffer components and concentrations and primer concentrations may be specified and incorporated into the algorithm, for example, an appropriate primer concentration is about 250 nM and appropriate buffer components are 50 mM sodium or potassium and 1.5 mM Mg²⁺.

One with ordinary skill in the art of design of amplification primers will recognize that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction. Moreover, a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event. (e.g., for example, a loop structure or a hairpin structure). The primers may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any of the primers listed in Table 2. Thus, in some embodiments, an extent of variation of 70% to 100%, or any range therewithin, of the sequence identity is possible relative to the specific primer sequences disclosed herein. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is identical to another 20 nucleobase primer having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.

Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, complementarity of primers with respect to the conserved priming regions of viral nucleic acid is between about 70% and about 75% 80%. In other embodiments, homology, sequence identity or complementarity, is between about 75% and about 80%. In yet other embodiments, homology, sequence identity or complementarity, is at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100%.

In some embodiments, the primers described herein comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range therewithin) sequence identity with the primer sequences specifically disclosed herein.

One with ordinary skill is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product of a corresponding bioagent identifying amplicon.

In one embodiment, the primers are at least 13 nucleobases in length. In another embodiment, the primers are less than 36 nucleobases in length.

In some embodiments, the oligonucleotide primers are 13 to 35 nucleobases in length (13 to 35 linked nucleotide residues). These embodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin. The methods disclosed herein contemplate use of both longer and shorter primers. Furthermore, the primers may also be linked to one or more other desired moieties, including, but not limited to, affinity groups, ligands, regions of nucleic acid that are not complementary to the nucleic acid to be amplified, labels, etc. Primers may also form hairpin structures. For example, hairpin primers may be used to amplify short target nucleic acid molecules. The presence of the hairpin may stabilize the amplification complex (see e.g., TAQMAN MicroRNA Assays, Applied Biosystems, Foster City, Calif.).

In some embodiments, any oligonucleotide primer pair may have one or both primers with less then 70% sequence homology with a corresponding member of any of the primer pairs of Table 2 if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon. In other embodiments, any oligonucleotide primer pair may have one or both primers with a length greater than 35 nucleobases if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon.

In some embodiments, the function of a given primer may be substituted by a combination of two or more primers segments that hybridize adjacent to each other or that are linked by a nucleic acid loop structure or linker which allows a polymerase to extend the two or more primers in an amplification reaction.

In some embodiments, the primer pairs used for obtaining bioagent identifying amplicons are the primer pairs of Table 2. In other embodiments, other combinations of primer pairs are possible by combining certain members of the forward primers with certain members of the reverse primers. An example can be seen in Table 2 for two primer pair combinations of forward primer 16S_EC_(—)789_(—)810_F (SEQ ID NO: 206), with the reverse primers 16S_EC_(—)880_(—)894_R (SEQ ID NO: 796), or 16S_EC_(—)882_(—)899_R or (SEQ ID NO: 818). Arriving at a favorable alternate combination of primers in a primer pair depends upon the properties of the primer pair, most notably the size of the bioagent identifying amplicon that would be produced by the primer pair, which preferably is between about 45 to about 200 nucleobases in length. Alternatively, a bioagent identifying amplicon longer than 200 nucleobases in length could be cleaved into smaller segments by cleavage reagents such as chemical reagents, or restriction enzymes, for example.

In some embodiments, the primers are configured to amplify nucleic acid of a bioagent to produce amplification products that can be measured by mass spectrometry and from whose molecular masses candidate base compositions can be readily calculated.

In some embodiments, any given primer comprises a modification comprising the addition of a non-templated T residue to the 5′ end of the primer (i.e., the added T residue does not necessarily hybridize to the nucleic acid being amplified). The addition of a non-templated T residue has an effect of minimizing the addition of non-templated adenosine residues as a result of the non-specific enzyme activity of Taq polymerase (Magnuson et al., Biotechniques, 1996, 21, 700-709), an occurrence which may lead to ambiguous results arising from molecular mass analysis.

In some embodiments, primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3^(rd) position) in the conserved regions among species is likely to occur in the third position of a DNA (or RNA) triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a “universal nucleobase.” For example, under this “wobble” pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal nucleobases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053-1056) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).

In some embodiments, to compensate for the somewhat weaker binding by the wobble base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs that bind with greater affinity than the unmodified nucleotide. Examples of these analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil (also known as propynylated thymine) which binds to adenine and 5-propynylcytosine and phenoxazines, including G-clamp, which binds to G. Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned and incorporated herein by reference in its entirety. Propynylated primers are described in U.S Pre-Grant Publication No. 2003-0170682, which is also commonly owned and incorporated herein by reference in its entirety. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each of which is incorporated herein by reference in its entirety. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which is incorporated herein by reference in its entirety.

In some embodiments, primer hybridization is enhanced using primers containing 5-propynyl deoxycytidine and deoxythymidine nucleotides. These modified primers offer increased affinity and base pairing selectivity.

In some embodiments, non-template primer tags are used to increase the melting temperature (T_(m)) of a primer-template duplex in order to improve amplification efficiency. A non-template tag is at least three consecutive A or T nucleotide residues on a primer which are not complementary to the template. In any given non-template tag, A can be replaced by C or G and T can also be replaced by C or G. Although Watson-Crick hybridization is not expected to occur for a non-template tag relative to the template, the extra hydrogen bond in a G-C pair relative to an A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency for subsequent cycles of amplification when the primers hybridize to strands synthesized in previous cycles.

In other embodiments, propynylated tags may be used in a manner similar to that of the non-template tag, wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer. In other embodiments, a primer contains a modified internucleoside linkage such as a phosphorothioate linkage, for example.

In some embodiments, the primers contain mass-modifying tags. Reducing the total number of possible base compositions of a nucleic acid of specific molecular weight provides a means of avoiding a persistent source of ambiguity in determination of base composition of amplification products. Addition of mass-modifying tags to certain nucleobases of a given primer will result in simplification of de novo determination of base composition of a given bioagent identifying amplicon from its molecular mass.

In some embodiments, the mass modified nucleobase comprises one or more of the following: for example, 7-deaza-2′-deoxyadenosine-5-triphosphate, 5-iodo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxycytidine-5′-triphosphate, 5-iodo-2′-deoxycytidine-5′-triphosphate, 5-hydroxy-2′-deoxyuridine-5′-triphosphate, 4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate, 5-fluoro-2′-deoxyuridine-5′-triphosphate, O6-methyl-2′-deoxyguanosine-5′-triphosphate, N2-methyl-2′-deoxyguanosine-5′-triphosphate, 8-oxo-2′-deoxyguanosine-5′-triphosphate or thiothymidine-5′-triphosphate. In some embodiments, the mass-modified nucleobase comprises ¹⁵N or ¹³C or both ¹⁵N and ¹³C.

In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with a plurality of primer pairs. The advantages of multiplexing are that fewer reaction containers (for example, wells of a 96- or 384-well plate) are needed for each molecular mass measurement, providing time, resource and cost savings because additional bioagent identification data can be obtained within a single analysis. Multiplex amplification methods are well known to those with ordinary skill and can be developed without undue experimentation. However, in some embodiments, one useful and non-obvious step in selecting a plurality candidate bioagent identifying amplicons for multiplex amplification is to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results. In some embodiments, a 10 Da difference in mass of two strands of one or more amplification products is sufficient to avoid overlap of mass spectral peaks.

In some embodiments, as an alternative to multiplex amplification, single amplification reactions can be pooled before analysis by mass spectrometry. In these embodiments, as for multiplex amplification embodiments, it is useful to select a plurality of candidate bioagent identifying amplicons to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results.

C Determination of Molecular Mass of Bioagent Identifying Amplicons

In some embodiments, the molecular mass of a given bioagent identifying amplicon is determined by mass spectrometry. Mass spectrometry has several advantages, not the least of which is high bandwidth characterized by the ability to separate (and isolate) many molecular peaks across a broad range of mass to charge ratio (m/z). Thus mass spectrometry is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.

In some embodiments, intact molecular ions are generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent identifying amplicon. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.

The mass detectors used in the methods described herein include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic sector, Q-TOF, and triple quadrupole.

D. Base Compositions of Bioagent Identifying Amplicons

Although the molecular mass of amplification products obtained using intelligent primers provides a means for identification of bioagents, conversion of molecular mass data to a base composition signature is useful for certain analyses. As used herein, “base composition” is the exact number of each nucleobase (A, T, C and G) determined from the molecular mass of a bioagent identifying amplicon. In some embodiments, a base composition provides an index of a specific organism. Base compositions can be calculated from known sequences of known bioagent identifying amplicons and can be experimentally determined by measuring the molecular mass of a given bioagent identifying amplicon, followed by determination of all possible base compositions which are consistent with the measured molecular mass within acceptable experimental error. The following example illustrates determination of base composition from an experimentally obtained molecular mass of a 46-mer amplification product originating at position 1337 of the 16S rRNA of Bacillus anthracis. The forward and reverse strands of the amplification product have measured molecular masses of 14208 and 14079 Da, respectively. The possible base compositions derived from the molecular masses of the forward and reverse strands for the B. anthracis products are listed in Table 1.

Table 1

Possible Base Compositions for B. anthracis 46mer Amplification Product Calc. Mass Base Calc. Mass Base Mass Error Composition Mass Error Composition Forward Forward of Forward Reverse Reverse of Reverse Strand Strand Strand Strand Strand Strand 14208.2935 0.079520 A1 G17 C10 14079.2624 0.080600 A0 G14 C13 T18 T19 14208.3160 0.056980 A1 G20 C15 14079.2849 0.058060 A0 G17 C18 T10 T11 14208.3386 0.034440 A1 G23 C20 T2 14079.3075 0.035520 A0 G20 C23 T3 14208.3074 0.065560 A6 G11 C3 T26 14079.2538 0.089180 A5 G5 C1 T35 14208.3300 0.043020 A6 G14 C8 T18 14079.2764 0.066640 A5 G8 C6 T27 14208.3525 0.020480 A6 G17 C13 14079.2989 0.044100 A5 G11 C11 T10 T19 14208.3751 0.002060 A6 G20 C18 T2 14079.3214 0.021560 A5 G14 C16 T11 14208.3439 0.029060 A11 G8 C1 T26 14079.3440 0.000980 A5 G17 C21 T3 14208.3665 0.006520 A11 G11 C6 14079.3129 0.030140 A10 G5 C4 T18 T27 14208.3890 0.016020 A11 G14 C11 14079.3354 0.007600 A10 G8 C9 T10 T19 14208.4116 0.038560 A11 G17 C16 14079.3579 0.014940 A10 G11 C14 T2 T11 14208.4030 0.029980 A16 G8 C4 T18 14079.3805 0.037480 A10 G14 C19 T3 14208.4255 0.052520 A16 G11 C9 14079.3494 0.006360 A15 G2 C2 T10 T27 14208.4481 0.075060 A16 G14 C14 14079.3719 0.028900 A15 G5 C7 T2 T19 14208.4395 0.066480 A21 G5 C2 T18 14079.3944 0.051440 A15 G8 C12 T11 14208.4620 0.089020 A21 G8 C7 T10 14079.4170 0.073980 A15 G11 C17 T3 — — — 14079.4084 0.065400 A20 G2 C5 T19 — — — 14079.4309 0.087940 A20 G5 C10 T13

Among the 16 possible base compositions for the forward strand and the 18 possible base compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary base compositions, which indicates the true base composition of the amplification product. It should be recognized that this logic is applicable for determination of base compositions of any bioagent identifying amplicon, regardless of the class of bioagent from which the corresponding amplification product was obtained.

In some embodiments, assignment of previously unobserved base compositions (also known as “true unknown base compositions”) to a given phylogeny can be accomplished via the use of pattern classifier model algorithms. Base compositions, like sequences, vary slightly from strain to strain within species, for example. In some embodiments, the pattern classifier model is the mutational probability model. On other embodiments, the pattern classifier is the polytope model. The mutational probability model and polytope model are both commonly owned and described in U.S. patent application Ser. No. 11/073,362 which is incorporated herein by reference in entirety.

In one embodiment, it is possible to manage this diversity by building “base composition probability clouds” around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A “pseudo four-dimensional plot” can be used to visualize the concept of base composition probability clouds. Optimal primer design requires optimal choice of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by a triangulation identification process using bioagent identifying amplicons not affected by overlap of base composition probability clouds.

In some embodiments, base composition probability clouds provide the means for screening potential primer pairs in order to avoid potential misclassifications of base compositions. In other embodiments, base composition probability clouds provide the means for predicting the identity of a bioagent whose assigned base composition was not previously observed and/or indexed in a bioagent identifying amplicon base composition database due to evolutionary transitions in its nucleic acid sequence. Thus, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition or sequence in order to make the measurement.

The methods disclosed herein provide bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to identify a given bioagent. Furthermore, the process of determination of a previously unknown base composition for a given bioagent (for example, in a case where sequence information is unavailable) has downstream utility by providing additional bioagent indexing information with which to populate base composition databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in base composition databases.

E. Triangulation Identification

In some cases, a molecular mass of a single bioagent identifying amplicon alone does not provide enough resolution to unambiguously identify a given bioagent. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by determining the molecular masses of a plurality of bioagent identifying amplicons selected within a plurality of housekeeping genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.

In some embodiments, the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR where multiple primers are employed in the same amplification reaction mixture, or PCR in multi-well plate format wherein a different and unique pair of primers is used in multiple wells containing otherwise identical reaction mixtures. Such multiplex and multi-well PCR methods are well known to those with ordinary skill in the arts of rapid throughput amplification of nucleic acids. In other related embodiments, one PCR reaction per well or container may be carried out, followed by an amplicon pooling step wherein the amplification products of different wells are combined in a single well or container which is then subjected to molecular mass analysis. The combination of pooled amplicons can be chosen such that the expected ranges of molecular masses of individual amplicons are not overlapping and thus will not complicate identification of signals.

F. Codon Base Composition Analysis

In some embodiments, one or more nucleotide substitutions within a codon of a gene of an infectious organism confer drug resistance upon an organism which can be determined by codon base composition analysis. The organism can be a bacterium, virus, fungus or protozoan.

In some embodiments, the amplification product containing the codon being analyzed is of a length of about 35 to about 200 nucleobases. The primers employed in obtaining the amplification product can hybridize to upstream and downstream sequences directly adjacent to the codon, or can hybridize to upstream and downstream sequences one or more sequence positions away from the codon. The primers may have between about 70% to 100% sequence complementarity with the sequence of the gene containing the codon being analyzed.

In some embodiments, the codon base composition analysis is undertaken

In some embodiments, the codon analysis is undertaken for the purpose of investigating genetic disease in an individual. In other embodiments, the codon analysis is undertaken for the purpose of investigating a drug resistance mutation or any other deleterious mutation in an infectious organism such as a bacterium, virus, fungus or protozoan. In some embodiments, the bioagent is a bacterium identified in a biological product.

In some embodiments, the molecular mass of an amplification product containing the codon being analyzed is measured by mass spectrometry. The mass spectrometry can be either electrospray (ESI) mass spectrometry or matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Time-of-flight (TOF) is an example of one mode of mass spectrometry compatible with the methods disclosed herein.

The methods disclosed herein can also be employed to determine the relative abundance of drug resistant strains of the organism being analyzed. Relative abundances can be calculated from amplitudes of mass spectral signals with relation to internal calibrants. In some embodiments, known quantities of internal amplification calibrants can be included in the amplification reactions and abundances of analyte amplification product estimated in relation to the known quantities of the calibrants.

In some embodiments, upon identification of one or more drug-resistant strains of an infectious organism infecting an individual, one or more alternative treatments can be devised to treat the individual.

G. Determination of the Quantity of a Bioagent

In some embodiments, the identity and quantity of an unknown bioagent can be determined using the process illustrated in FIG. 2. Primers (500) and a known quantity of a calibration polynucleotide (505) are added to a sample containing nucleic acid of an unknown bioagent. The total nucleic acid in the sample is then subjected to an amplification reaction (510) to obtain amplification products. The molecular masses of amplification products are determined (515) from which are obtained molecular mass and abundance data. The molecular mass of the bioagent identifying amplicon (520) provides the means for its identification (525) and the molecular mass of the calibration amplicon obtained from the calibration polynucleotide (530) provides the means for its identification (535). The abundance data of the bioagent identifying amplicon is recorded (540) and the abundance data for the calibration data is recorded (545), both of which are used in a calculation (550) which determines the quantity of unknown bioagent in the sample.

A sample comprising an unknown bioagent is contacted with a pair of primers that provide the means for amplification of nucleic acid from the bioagent, and a known quantity of a polynucleotide that comprises a calibration sequence. The nucleic acids of the bioagent and of the calibration sequence are amplified and the rate of amplification is reasonably assumed to be similar for the nucleic acid of the bioagent and of the calibration sequence. The amplification reaction then produces two amplification products: a bioagent identifying amplicon and a calibration amplicon. The bioagent identifying amplicon and the calibration amplicon should be distinguishable by molecular mass while being amplified at essentially the same rate. Effecting differential molecular masses can be accomplished by choosing as a calibration sequence, a representative bioagent identifying amplicon (from a specific species of bioagent) and performing, for example, a 2-8 nucleobase deletion or insertion within the variable region between the two priming sites. The amplified sample containing the bioagent identifying amplicon and the calibration amplicon is then subjected to molecular mass analysis by mass spectrometry, for example. The resulting molecular mass analysis of the nucleic acid of the bioagent and of the calibration sequence provides molecular mass data and abundance data for the nucleic acid of the bioagent and of the calibration sequence. The molecular mass data obtained for the nucleic acid of the bioagent enables identification of the unknown bioagent and the abundance data enables calculation of the quantity of the bioagent, based on the knowledge of the quantity of calibration polynucleotide contacted with the sample.

In some embodiments, construction of a standard curve where the amount of calibration polynucleotide spiked into the sample is varied provides additional resolution and improved confidence for the determination of the quantity of bioagent in the sample. The use of standard curves for analytical determination of molecular quantities is well known to one with ordinary skill and can be performed without undue experimentation.

In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with multiple primer pairs which also amplify the corresponding standard calibration sequences. In this or other embodiments, the standard calibration sequences are optionally included within a single vector which functions as the calibration polynucleotide. Multiplex amplification methods are well known to those with ordinary skill and can be performed without undue experimentation.

In some embodiments, the calibrant polynucleotide is used as an internal positive control to confirm that amplification conditions and subsequent analysis steps are successful in producing a measurable amplicon. Even in the absence of copies of the genome of a bioagent, the calibration polynucleotide should give rise to a calibration amplicon. Failure to produce a measurable calibration amplicon indicates a failure of amplification or subsequent analysis step such as amplicon purification or molecular mass determination. Reaching a conclusion that such failures have occurred is in itself, a useful event.

In some embodiments, the calibration sequence is comprised of DNA. In some embodiments, the calibration sequence is comprised of RNA.

In some embodiments, the calibration sequence is inserted into a vector that itself functions as the calibration polynucleotide. In some embodiments, more than one calibration sequence is inserted into the vector that functions as the calibration polynucleotide. Such a calibration polynucleotide is herein termed a “combination calibration polynucleotide.” The process of inserting polynucleotides into vectors is routine to those skilled in the art and can be accomplished without undue experimentation. Thus, it should be recognized that the calibration method should not be limited to the embodiments described herein. The calibration method can be applied for determination of the quantity of any bioagent identifying amplicon when an appropriate standard calibrant polynucleotide sequence is designed and used. The process of choosing an appropriate vector for insertion of a calibrant is also a routine operation that can be accomplished by one with ordinary skill without undue experimentation.

H. Identification of Bacteria

In other embodiments, the primer pairs produce bioagent identifying amplicons within stable and highly conserved regions of bacteria. The advantage to characterization of an amplicon defined by priming regions that fall within a highly conserved region is that there is a low probability that the region will evolve past the point of primer recognition, in which case, the primer hybridization of the amplification step would fail. Such a primer set is thus useful as a broad range survey-type primer. In another embodiment, the intelligent primers produce bioagent identifying amplicons including a region which evolves more quickly than the stable region described above. The advantage of characterization bioagent identifying amplicon corresponding to an evolving genomic region is that it is useful for distinguishing emerging strain variants or the presence of virulence genes, drug resistance genes, or codon mutations that induce drug resistance.

The methods disclosed herein have significant advantages as a platform for identification of diseases caused by emerging bacterial strains such as, for example, drug-resistant strains of Staphylococcus aureus. The methods disclosed herein eliminate the need for prior knowledge of bioagent sequence to generate hybridization probes. This is possible because the methods are not confounded by naturally occurring evolutionary variations occurring in the sequence acting as the template for production of the bioagent identifying amplicon. Measurement of molecular mass and determination of base composition is accomplished in an unbiased manner without sequence prejudice.

Another embodiment also provides a means of tracking the spread of a bacterium, such as a particular drug-resistant strain when a plurality of samples obtained from different locations are analyzed by the methods described above in an epidemiological setting. In one embodiment, a plurality of samples from a plurality of different locations is analyzed with primer pairs which produce bioagent identifying amplicons, a subset of which contains a specific drug-resistant bacterial strain. The corresponding locations of the members of the drug-resistant strain subset indicate the spread of the specific drug-resistant strain to the corresponding locations.

Another embodiment provides the means of identifying a sepsis-causing bacterium. The sepsis-causing bacterium is identified in samples including, but not limited to blood.

Sepsis-causing bacteria include, but are not limited to the following bacteria: Prevotella denticola, Porphyromonas gingivalis, Borrelia burgdorferi, Mycobacterium tuburculosis, Mycobacterium fortuitum, Corynebacteriumjeikeium, Propionibacterium acnes, Mycoplasma pneumoniae, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus mitis, Streptococcus pyogenes, Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus coagulase-negative, Staphylococcus epidermis, Staphylococcus hemolyticus, Campylobacter jejuni, Bordatella pertussis, Burkholderia cepacia, Legionella pneumophila, Acinetobacter baumannii, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, Aeromonas hydrophila, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Moxarella catarrhalis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Pantoea agglomerans, Bartonella henselae, Stenotrophomonas maltophila, Actinobacillus actinomycetemcomitans, Haemophilus influenzae, Escherichia coli, Klebsiella oxytoca, Serratia marcescens, and Yersinia enterocolitica.

In some embodiments, identification of a sepsis-causing bacterium provides the information required to choose an antibiotic with which to treat an individual infected with the sepsis-causing bacterium and treating the individual with the antibiotic. Treatment of humans with antibiotics is well known to medical practitioners with ordinary skill.

I. Kits

Also provided are kits for carrying out the methods described herein. In some embodiments, the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon. In some embodiments, the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs. In some embodiments, the kit may comprise one or more primer pairs recited in Table 2.

In some embodiments, the kit comprises one or more broad range survey primer(s), division wide primer(s), or drill-down primer(s), or any combination thereof. If a given problem involves identification of a specific bioagent, the solution to the problem may require the selection of a particular combination of primers to provide the solution to the problem. A kit may be designed so as to comprise particular primer pairs for identification of a particular bioagent. A drill-down kit may be used, for example, to distinguish different genotypes or strains, drug-resistant, or otherwise. In some embodiments, the primer pair components of any of these kits may be additionally combined to comprise additional combinations of broad range survey primers and division-wide primers so as to be able to identify a bacterium.

In some embodiments, the kit contains standardized calibration polynucleotides for use as internal amplification calibrants. Internal calibrants are described in commonly owned PCT Publication Number WO 2005/098047 which is incorporated herein by reference in its entirety.

In some embodiments, the kit comprises a sufficient quantity of reverse transcriptase (if RNA is to be analyzed for example), a DNA polymerase, suitable nucleoside triphosphates (including alternative dNTPs such as inosine or modified dNTPs such as the 5-propynyl pyrimidines or any dNTP containing molecular mass-modifying tags such as those described above), a DNA ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described above. A kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like. A kit may also comprise reagents or other materials for isolating bioagent nucleic acid or bioagent identifying amplicons from amplification, including, for example, detergents, solvents, or ion exchange resins which may be linked to magnetic beads. A kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit.

Some embodiments are kits that contain one or more survey bacterial primer pairs represented by primer pair compositions wherein each member of each pair of primers has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by any of the primer pairs of Table 5. The survey primer pairs may include broad range primer pairs which hybridize to ribosomal RNA, and may also include division-wide primer pairs which hybridize to housekeeping genes such as rplB, tufB, rpoB, rpoC, valS, and infB, for example.

In some embodiments, a kit may contain one or more survey bacterial primer pairs and one or more triangulation genotyping analysis primer pairs such as the primer pairs of Tables 8, 12, 14, 19, 21, 23, or 24. In some embodiments, the kit may represent a less expansive genotyping analysis but include triangulation genotyping analysis primer pairs for more than one genus or species of bacteria. For example, a kit for surveying nosocomial infections at a health care facility may include, for example, one or more broad range survey primer pairs, one or more division wide primer pairs, one or more Acinetobacter baumannii triangulation genotyping analysis primer pairs and one or more Staphylococcus aureus triangulation genotyping analysis primer pairs. One with ordinary skill will be capable of analyzing in silico amplification data to determine which primer pairs will be able to provide optimal identification resolution for the bacterial bioagents of interest.

In some embodiments, a kit may be assembled for identification of strains of bacteria involved in contamination of food. An example of such a kit embodiment is a kit comprising one or more bacterial survey primer pairs of Table 5 with one or more triangulation genotyping analysis primer pairs of Table 12 which provide strain resolving capabilities for identification of specific strains of Campylobacter jejuni.

In some embodiments, a kit may be assembled for identification of sepsis-causing bacteria. An example of such a kit embodiment is a kit comprising one or more of the primer pairs of Table 25 which provide for a broad survey of sepsis-causing bacteria.

Some embodiments of the kits are 96-well or 384-well plates with a plurality of wells containing any or all of the following components: dNTPs, buffer salts, Mg²⁺, betaine, and primer pairs. In some embodiments, a polymerase is also included in the plurality of wells of the 96-well or 384-well plates.

Some embodiments of the kit contain instructions for PCR and mass spectrometry analysis of amplification products obtained using the primer pairs of the kits.

Some embodiments of the kit include a barcode which uniquely identifies the kit and the components contained therein according to production lots and may also include any other information relative to the components such as concentrations, storage temperatures, etc. The barcode may also include analysis information to be read by optical barcode readers and sent to a computer controlling amplification, purification and mass spectrometric measurements. In some embodiments, the barcode provides access to a subset of base compositions in a base composition database which is in digital communication with base composition analysis software such that a base composition measured with primer pairs from a given kit can be compared with known base compositions of bioagent identifying amplicons defined by the primer pairs of that kit.

In some embodiments, the kit contains a database of base compositions of bioagent identifying amplicons defined by the primer pairs of the kit. The database is stored on a convenient computer readable medium such as a compact disk or USB drive, for example.

In some embodiments, the kit includes a computer program stored on a computer formatted medium (such as a compact disk or portable USB disk drive, for example) comprising instructions which direct a processor to analyze data obtained from the use of the primer pairs disclosed herein. The instructions of the software transform data related to amplification products into a molecular mass or base composition which is a useful concrete and tangible result used in identification and/or classification of bioagents. In some embodiments, the kits contain all of the reagents sufficient to carry out one or more of the methods described herein.

While the present invention has been described with specificity in accordance with certain of its embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.

EXAMPLES Example 1 Design and Validation of Primers that Define Bioagent Identifying Amplicons for Identification of Bacteria

For design of primers that define bacterial bioagent identifying amplicons, a series of bacterial genome segment sequences were obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 200 nucleotides in length and distinguish subgroups and/or individual strains from each other by their molecular masses or base compositions. A typical process shown in FIG. 1 is employed for this type of analysis.

A database of expected base compositions for each primer region was generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs.

Table 2 represents a collection of primers (sorted by primer pair number) designed to identify bacteria using the methods described herein. The primer pair number is an in-house database index number. Primer sites were identified on segments of genes, such as, for example, the 16S rRNA gene. The forward or reverse primer name shown in Table 2 indicates the gene region of the bacterial genome to which the primer hybridizes relative to a reference sequence. In Table 2, for example, the forward primer name 16 S_EC_(—)1077_(—)1106_F indicates that the forward primer (_F) hybridizes to residues 1077-1106 of the reference sequence represented by a sequence extraction of coordinates 4033120..4034661 from GenBank gi number 16127994 (as indicated in Table 3). As an additional example: the forward primer name BONTA_X52066_(—)450_(—)473 indicates that the primer hybridizes to residues 450-437 of the gene encoding Clostridium botulinum neurotoxin type A (BoNT/A) represented by GenBank Accession No. X52066 (primer pair name codes appearing in Table 2 are defined in Table 3. One with ordinary skill will know how to obtain individual gene sequences or portions thereof from genomic sequences present in GenBank. In Table 2, Tp=5-propynyluracil; Cp=5-propynylcytosine; *=phosphorothioate linkage; I=inosine. T. GenBank Accession Numbers for reference sequences of bacteria are shown in Table 3 (below). In some cases, the reference sequences are extractions from bacterial genomic sequences or complements thereof.

TABLE 2 Primer Pairs for Identification of Bacteria Pri- For- Re- mer ward verse Pair SEQ SEQ Num- ID ID ber Forward Primer Name Forward Sequence NO: Reverse Primer Name Reverse Sequence NO:    1 16S_EC_1077_1106_F GTGAGATGTTGGGTTAAGTCCC 134 16S_EC_1175_119_R GACGTCATCCCCACCTTCCTC 809 GTAACGAG    2 16S_EC_1082_1106_F ATGTTGGGTTAAGTCCCGCAAC 38 16S_EC_1175_1197_R TTGACGTCATCCCCACCTTCC 1398 GAG TC    3 16S_EC_1090_1111_F TTAAGTCCCGCAACGATCGCAA 651 16S_EC_1175_1196_R TGACGTCATCCCCACCTTCCTC 1159    4 16S_EC_1222_1241_F GCTACACACGTGCTACAATG 114 16S_EC_1303_1323_R CGAGTTGCAGACTGCGATCCG 787    5 16S_EC_1332_1353_F AAGTCGGAATCGCTAGTAATCG 10 16S_EC_1389_1407_R GACGGGCGGTGTGTACAAG 806    6 16S_EC_30_54_F TGAACGCTGGTGGCATGCTTAA 429 16S_EC_105_126_R TACGCATTACTCACCCGTCCGC 897 CAC    7 16S_EC_38_64_F GTGGCATGCCTAATACATGCAA 136 16ES_EC_101_120_R TTACTCACCCGTCCGCCGCT 1365 GTCG    8 16S_EC_49_68_F TAACACATGCAAGTCGAACG 152 16S_EC_104_120_R TTACTCACCCGTCCGCC 1364    9 16S_EC_683_700_F GTGTAGCGGTGAAATGCG 137 16S_EC_774_795_R GTATCTAATCCTGTTTGCTCCC 839   10 16S_EC_713_732_F AGAACACCGATGGCGAAGGC 21 16S_EC_789_809 R CGTGGACTACCAGGGTATCTA 798   11 16S_EC_785_806_F GGATTAGAGACCCTGGTAGTCC 118 16S_EC_880_897_R GGCCGTACTCCCCAGGCG 830   12 16S_EC_785_810_F GGATTAGATACCCTGGTAGTCC 119 16S_EC_880_897_2_R GGCCGTACTCCCCAGGCG 830 ACGC   13 16S_EC_789_810_F TAGATACCCTGGTAGTCCACGC 206 16S_EC_880_894_R CGTACTCCCCAGGCG 796   14 16S_EC_960_981_F TTCGATGCAACGCGAAGAACCT 672 16S_EC_1054_1073_R ACGAGCTGACGACAGCCATG 735   15 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078_R ACGACACGAGCTGACGAC 734   16 23S_EC_1826_1843_F CTGACACCTGCCCGGTGC 80 23S_EC_1906_1924_R GACCGTTATAGTTACGGCC 805   17 23S_EC_2645_2669_F TCTGTCCCTAGTACGAGAGGAC 408 23S_EC_2744_2761_R TGCTTAGATGCTTTCAGC 1252 CGG   18 23S_EC_2645_2669_2_ CTGTCCCTAGTACGAGAGGACC 83 23S_EC_2751_2767_R GTTTCATGCTTAGATGCTTTC 846 F GG AGC   19 23S_EC_493 518_F GGGGAGTGAAAGAGATCCTGAA 125 23S_EC_551_571_R ACAAAAGGTACGCCGTCACCC 717 ACCG   20 23S_EC_493_518_2_F GGGGAGTGAAAGAGATCCTGAA 125 23S_EC_551_571_2_R ACAAAAGGCACGCCATCACCC 716 ACCG   21 23S_EC_971_992_F CGAGAGGGAAACAACCCAGACC 66 23S_EC_1059_1077_R TGGCTGCTTCTAAGCCAAC 1282   22 CAPC_BA_104_131_F GTTATTTAGCACTCGTTTTTAA 139 CAPC_BA_180_205_R TGAATCTTGAAACACCATACG 1150 TCAGCC TAACG   23 CAPC_BA_114_133_F ACTCGTTTTTAATCAGCCCG 20 CAPC_BA_185_205_R TGAATCTTGAAACACCATACG 1149   24 CAPC_BA_274_303_F GATTATTGTTATCCTGTTATGC 109 CAPC_BA_349_376_R GTAACCCTTGTCTTTGAATTG 837 CATTTGAG TATTTGC   25 CAPC_BA_276_296_F TTATTGTTATCCTGTTATGCC 663 CAPC_BA_358_377_R GGTAACCCTTGTCTTTGAAT 834   26 CAPC_BA_281_301_F GTTATCCTGTTATGCCATTTG 138 CAPC_BA_361_378_R TGGTAACCCTTGTCTTTG 1298   27 CAPC_BA_315_334_F CCGTGGTATTGGAGTTATTG 59 CAPC_BA_361_378_R TGGTAACCCTTGTCTTTG 1298   28 CYA_BA_1055_1072_F GAAAGAGTTCGGATTGGG 92 CYA_BA_1112_1130_R TGTTGACCATGCTTCTTAG 1352   29 CYA_BA_1349_1370_F ACAACGAAGTACAATACAAGAC 12 CYA_BA_1447_1426_R CTTCTACATTTTTAGCCATCAC 800   30 CYA_BA_1353_1379_F CGAAGTACAATACAAGACAAAA 64 CYA_BA_1448_1467_R TGTTAACGGCTTCAAGACCC 1342 GAAGG   31 CYA_BA_1359_1379_F ACAATACAAGACAAAAGAAGG 13 CYA_BA_1447_1461_R CGGCTTCAAGACCCC 794   32 CYA_BA_914_937_F CAGGTTTAGTACCAGAACATGC 53 CYA_BA_999_1026_R ACCACTTTTAATAAGGTTTGT 728 AG AGCTAAC   33 CYA_BA_916_935_F GGTTTAGTACCAGAACATGC 131 CYA_BA_1003_1025_R CCACTTTTAATAAGGTTTGTA 768 GC   34 INFB_EC_1365_1393_F TGCTCGTGGTGCACAAGTAACG 524 INFB_EC_1439_1467_R TGCTGCTTTCGCATGGTTAAT 1248 GATATTA TGCTTCAA   35 LEF_BA_1033_1052_F TCAAGAAGAAAAAGAGC 254 LEF_BA_1119_1135_R GAATATCAATTTGTAGC 803   36 LEF_BA_1036_1066_F CAAGAAGAAAAAGAGCTTCTAA 44 LEF_BA_1119_1149_R AGATAAAGAATCACGAATATC 745 AAAGAATAC AATTTGTAGC   37 LEF BA_756_781_F AGCTTTTGCATATTATATCGAG 26 LEF_BA_843_872_R TCTTCCAAGGATAGATTTATT 1135 CCAC TCTTGTTCG   38 LEF_BA_758_778_F CTTTTGCATATTATATCGAGC 90 LEF_BA_843_865_R AGGATAGATTTATTTCTTGTT 748 CG   39 LEF_BA_795_813_F TTTACAGCTTTATGCACCG 700 LEF_BA_883_900_R TCTTGACAGCATCCGTTG 1140   40 LEF_BA_883_899_F CAACGGATGCTGGCAAG 43 LEF_BA_939_958_R CAGATAAAGAATCGCTCCAG 762   41 PAG_BA_122_142_F CAGAATCAAGTTCCCAGGGG 49 PAG_BA_190_209_R CCTGTAGTAGAAGAGGTAAC 781   42 PAG_BA_123_145_F AGAATCAAGTTCCCAGGGGTTAC 22 PAG_BA_187_210_R CCCTGTAGTAGAAGAGGTAAC 774 CAC   43 PAG_BA_269_287_F AATCTGCTATTTGGTCAGG 11 PAG_BA_326_344_R TGATTATCAGCGGAAGTAG 1186   44 PAG_BA_655_675_F GAAGGATATACGGTTGATGTC 93 PAG_BA_755_772_R CCGTGCTCCATTTTTCAG 778   45 PAG_BA_753_772_F TCCTGAAAAATGGAGCACGG 341 PAG_BA_849_868_R TCGGATAAGCTGCCACAAGG 1089   46 PAG_BA_763_781_F TGGAGCACGGCTTCTGATC 552 PAG_BA_849_868_R TCGGATAAGCTGCCACAAGG 1089   47 RPOC_EC_1018_1045_F CAAAACTTATTAGGTAAGCGTG 39 RPOC_EC_1095_1124_2_R TCAAGCGCCATTTCTTTTGGT 959 TTGACT AAACCACAT   48 RPOC_EC_1018_1045_ CAAAACTTATTAGGTAAGCGTG 39 RPOC_EC_1095_1124_2_ TCAAGCGCCATCTCTTTCGGT 958 2_F TTGACT R AATCCACAT   49 RPOC_EC_114_140_F TAAGAAGCCGGAAACCATCAAC 158 RPOC_EC_213_232_R GGCGCTTGTACTTACCGCAC 831 TACCG   50 RPOC_EC_2178_2196_F TGATTCTGGTGCCCGTGGT 478 RPOC_EC_2225_2246_R TTGGCCATCAGGCCACGCATAC 1414   51 RPOC_EC_2178_2196_ TGATTCCGGTGCCCGTGGT 477 RPOC_EC_2225_2246_2_R TTGGCCATCAGACCACGCATAC 1413 2_F   52 RPOC_EC_2218_2241_F CTGGCAGGTATGCGTGGTCTGA 81 RPOC_EC_2313_2337_R CGCACCGTGGGTTGAGATGAA 790 TG GTAC   53 RPOC_EC_2218_2241_ CTTGCTGGTATGCGTGGTCTGA 86 RPOC_EC_2313_2337_2_R CGCACCATGCGTAGAGATGAA 789 2_F TG GTAC   54 RPOC_EC_808_833_F CGTCGGGTGATTAACCGTAACA 75 RPOC_EC_865_889_R GTTTTTCGTTGCGTACGATGA 847 ACCG TGTC   55 RPOC_EC_808_833_2_F CGTCGTGTAATTAACCGTAACA 76 RPOC_EC_865_891_R ACGTTTTTCGTTTTGAACGAT 741 ACCG AATGCT   56 RPOC_EC_993_1019_F CAAAGGTAAGCAAGGTCGTTTC 41 RPOC_EC_1036_1059_R CGAACGGCCTGAGTAGTCAAC 785 CGTCA ACG   57 RPOC_EC_993_1019_2_ CAAAGGTAAGCAAGGACGTTTC 40 RPOC_EC_1036_1059_2_R CGAACGGCCAGAGTAGTCAAC 784 F CGTCA ACG   58 SSPE_BA_115_137_F CAAGCAAACGCACAATCAGAAG 45 SSPE_BA_197_222_R TGCACGTCTGTTTCAGTTGCA 1201 C AATTC   59 TUFB_EC_239_259_F TAGACTGCCCAGGACACGCTG 204 TUFB_EC_283_303_R GCCGTCCATCTGAGCAGCACC 815   60 TUFB_EC_239_259_2_F TTGACTGCCCAGGTCACGCTG 678 TUFB_EC_283_303_2_R GCCGTCCATTTGAGCAGCACC 816   61 TUFB_EC_976_1000_F AACTACCGTCCGCAGTTCTACT 4 TUFB_EC_1045_1068_R GTTGTCGCCAGGCATAACCAT 845 TCC TTC   62 TUFB_EC_976_1000_ AACTACCGTCCTCAGTTCTACT 5 TUFB_EC_1045_1068_2_R GTTGTCACCAGGCATTACCAT 844 2_F TCC TTC   63 TUFB_EC_985_1012_F CCACAGTTCTACTTCCGTACTA 56 TUFB_EC_1033_1062_R TCCAGGCATTACCATTTCTAC 1006 CTGACG TCCTTCTGG   66 RPLB_EC_650_679_F GACCTACAGTAAGAGGTTCTGT 98 RPLB_EC_739_762_R TCCAAGTGCTGGTTTACCCCA 999 AATGAACC TGG   67 RPLB_EC_688_710_F CATCCACACGGTGGTGGTGAAG 54 RPLB_EC_736_757_R GTGCTGGTTTACCCCATGGAGT 842 G   68 RPOC_EC_1036_1060_F CGTGTTGACTATTCGGGGCGTT 78 RPOC_EC_1097_1126_R ATTCAAGAGCCATTTCTTTTG 754 CAG GTAAACCAC   69 RPOB_EC_3762_3790_F TCAACAACCTCTTGGAGGTAAA 248 RPOB_EC_3836_3865_R TTTCTTGAAGAGTATGAGCTG 1435 GCTCAGT CTCCGTAAG   70 RPLB_EC_688_710_F CATCCACACGGTGGTGGTGAAG 54 RPLB_EC_743_771_R TGTTTTGTATCCAAGTGCTGG 1356 G TTTACCCC   71 VALS_EC_1105_1124_F CGTGGCGGCGTGGTTATCGA 77 VALS_EC_1195_1218_R CGGTACGAACTGGATGTCGCC 795 GTT   72 RPOB_EC_1845_1866_F TATCGCTCAGGCGAACTCCAAC 233 RPOB_EC_1909_1929_R GCTGGATTCGCCTTTGCTACG 825   73 RPLB_EC_669_698_F TGTAATGAACCCTAATGACCAT 623 RPLB_EC_735_761_R CCAAGTGCTGGTTTACCCCAT 767 CCACACGG GGAGTA   74 RPLB_EC_671_700_F TAATGAACCCTAATGACCATCC 169 RPLB_EC_737_762_R TCCAAGTGCTGGTTTACCCCA 1000 ACACGGTG TGGAG   75 SP101_SPET11_1_29_F AACCTTAATTGGAAAGAAACCC 2 SP101_SPET11_92_116_R CCTACCCAACGTTCACCAAGG 779 AAGAAGT GCAG   76 SP101_SPET11_118_ GCTGGTGAAAATAACCCAGATG 115 SP101_SPET11_213_238_ TGTGGCCGATTTCACCACCTG 1340 147_F TCGTCTTC R CTCCT   77 SP101_SPET11_216_ AGCAGGTGGTGAAATCGGCCAC 24 SP101_SPET11_308_333_ TGCCACTTTGACAACTCCTGT 1209 243_F ATGATT R TGCTG   78 SP101_SPET11_266_ CTTGTACTTGTGGCTCACACGG 89 SP101_SPET11_355_380_ GCTGCTTTGATGGCTGAATCC 824 295_F CTGTTTGG R CCTTC   79 SP101_SPET11_322_ GTCAAAGTGGCACGTTTACTGG 132 SP101_SPET11_423_441_ ATCCCCTGCTTCTGCTGCC 753 344_F C R   80 SP101_SPET11_358_ GGGGATTCAGCCATCAAAGCAG 126 SP101_SPET11_448_473_ CCAACCTTTTCCACAACAGAA 766 387_F CTATTGAC R TCAGC   81 SP101_SPET11_600_ CCTTACTTCGAACTATGAATCT 62 SP101_SPET11_686_714_ CCCATTTTTTCACGCATGCTG 772 629_F TTTGGAAG R AAAATATC   82 SP101_SPET11_658_ GGGGATTGATATCACCGATAAG 127 SP101_SPET11_756_784_ GATTGGCGATAAAGTGATATT 813 684_F AAGAA R TTCTAAAA   83 SP101_SPET11_776_ TCGCCAATCAAAACTAAGGGAA 364 SP101_SPET11_871_896_ GCCCACCAGAAAGACTAGCAG 814 801_F TGGC R GATAA   84 SP101_SPET11_893_ GGGCAACAGCAGCGGATTGCGA 123 SP101_SPET11_988_ CATGACAGCCAAGACCTCACC 763 921_F TTGCGCG 1012_R CACC   85 SP101_SPET11_1154_ CAATACCGCAACAGCGGTGGCT 47 SP101_SPET11_1251_ GACCCCAACCTGGCCTTTTGT 804 1179_F TGGG 1277_R CGTTGA   86 SP101_SPET11_1314_ CGCAAAAAAATCCAGCTATTAG 68 SP101_SPET11_1403_ AAACTATTTTTTTAGCTATAC 711 1336_F C 1432_R TCGAACAC   87 SP101_SPET11_1408_ CGAGTATAGCTAAAAAAATAGT 67 SP101_SPET11_1486_ GGATAATTGGTCGTAACAAGG 828 1437_F TTATGACA 1515_R GATAGTGAG   88 SP101_SPET11_1688_ CCTATATTAATCGTTTACAGAA 60 SP101_SPET11_1783_ ATATGATTATCATTGAACTGC 752 1716_F ACTGGCT 1808_R GGCCG   89 SP101_SPET11_1711_ CTGGCTAAAACTTTGGCAACGG 82 SP101_SPET11_1808_ GCGTGACGACCTTCTTGAATT 821 1733_F T 1835_R GTAATCA   90 SP101_SPET11_1807_ ATGATTACAATTCAAGAAGGTC 33 SP101_SPET11_1901_ TTGGACCTGTAATCAGCTGAA 1412 1835_F GTCACGC 1927_R TACTGG   91 SP101_SPET11_1967_ TAACGGTTATCATGGCCCAGAT 155 SP101_SPET11_2062_ ATTGCCCAGAAATCAAATCATC 755 1991_F GGG 2083_R   92 SP101_SPET11_2260_ CAGAGACCGTTTTATCCTATCA 50 SP101_SPET11_2375_ TCTGGGTGACCTGGTGTTTTA 1131 2283_F GC 2397_R GA   93 SP101_SPET11_2375_ TCTAAAACACCAGGTCACCCAG 390 SP101_SPET11_2470_ AGCTGCTAGATGAGCTTCTGC 747 2399_F AAG 2497_R CATGGCC   94 SP101_SPET11_2468_ ATGGCCATGGCAGAAGCTCA 35 SP101_SPET11_2543_ CCATAAGGTCACCGTCACCAT 770 2487_F 2570_R TCAAAGC   95 SP101_SPET11_2961_ ACCATGACAGAAGGCATTTTGA 15 SP101_SPET11_3023_ GGAATTTACCAGCGATAGACA 827 2984_F CA 3045_R CC   96 SP101_SPET11_3075_ GATGACTTTTTAGCTAATGGTC 108 SP101_SPET11_3168_ AATCGACGACCATCTTGGAAA 715 3103_F AGGCAGC 3196_R GATTTCTC   97 SP101_SPET11_3386_ AGCGTAAAGGTGAACCTT 25 SP101_SPET11_3480_ CCAGCAGTTACTGTCCCCTCA 769 3403_F 3506_R TCTTTG   98 SP101_SPET11_3511_ GCTTCAGGAATCAATGATGGAG 116 SP101_SPET11_3605_ GGGTCTACACCTGCACTTGCA 832 3535_F CAG 3629_R TAAC  111 RPOB_EC_3775_3803_F CTTGGAGGTAAGTCTCATTTTG 87 RPOB_EC_3829_3858_R CGTATAAGCTGCACCATAAGC 797 GTGGGCA TTGTAATGC  112 VALS_EC_1833_1850_F CGACGCGCTGCGCTTCAC 65 VALS_EC_1920_1943_R GCGTTCCACAGCTTGTTGCAG 822 AAG  113 RPOB_EC_1336_1353_F GACCACCTCGGCAACCGT 97 RPOB_EC_1438_1455_R TTCGCTCTCGGCCTGGCC 1386  114 TUFB_EC_225_251_F GCACTATGCACACGTAGATTGT 111 TUFB_EC_284_309_R TATAGCACCATCCATCTGAGC 930 CCTGG GGCAC  115 DNAK_EC_428_449_F CGGCGTACTTCAACGACAGCCA 72 DNAK_EC_503_522_R CGCGGTCGGCTCGTTGATGA 792  116 VALS_EC_1920_1943_F CTTCTGCAACAAGCTGTGGAAC 85 VALS_EC_1948_1970_R TCGCAGTTCATCAGCACGAAG 1075 GC CG  117 TUFB_EC_757_774_F AAGACGACCTGCACGGGC 6 TUFB_EC_849_867_R GCGCTCCACGTCTTCACGC 819  118 23S_EC_2646_2667_F CTGTTCTTAGTACGAGAGGACC 84 23S_EC_2745_2765_R TTCGTGCTTAGATGCTTTCAG 1389  119 16S_EC_969_985_1P_F ACGCGAAGAACCTTACpC 19 16S_EC_1061_1078_2P_R ACGACACGAGCpTpGACGAC 733  120 16S_EC_972_985_2P_F CGAAGAACpCpTTACC 63 16S_EC_1064_1075_2P_R ACACGAGCpTpGAC 727  121 16S_EC_972_985_F CGAAGAACCTTACC 63 16S_EC_1064_1075_R ACACGAGCTGAC 727  122 TRNA_ILE- CCTGATAAGGGTGAGGTCG 61 23S_EC_40_59_R ACGTCCTTCATCGCCTCTGA 740 RRNH_EC_32_50.2_F  123 23S_EC_−7_15_F GTTGTGAGGTTAAGCGACTAAG 140 23S_EC_430_450_R CTATCGGTCAGTCAGGAGTAT 799  124 23S_EC_−7_15_F GTTGTGAGGTTAAGCGACTAAG 141 23S_EC_891_910_R TTGCATCGGGTTGGTAAGTC 1403  125 23S_EC_430_450_F ATACTCCTGACTGACCGATAG 30 23S_EC_1424_1442_R AACATAGCCTTCTCCGTCC 712  126 23S_EC_891_910_F GACTTACCAACCCGATGCAA 100 23S_EC_1908_1931_R TACCTTAGGACCGTTATAGTT 893 ACG  127 23S_EC_1424_1442_F GGACGGAGAAGGCTATGTT 117 23S_EC_2475_2494_R CCAAACACCGCCGTCGATAT 765  128 23S_EC_1908_1931_F CGTAACTATAACGGTCCTAAGG 73 23S_EC_2833_2852_R GCTTACACACCCGGCCTATC 826 TA  129 23S_EC_2475_2494_F ATATCGACGGCGGTGTTTGG 31 TRNA_ASP- GCGTGACAGGCAGGTATTC 820 RRNH_EC_23_41.2_R  131 16S_EC_−60_−39_F AGTCTCAAGAGTGAACACGTAA 28 16S_EC_508_525_R GCTGCTGGCACGGAGTTA 823  132 16S_EC_326_345_F GACACGGTCCAGACTCCTAC 95 16S_EC_1041_1058_R CCATGCAGCACCTGTCTC 771  133 16S_EC_705_724_F GATCTGGAGGAATACCGGTG 107 16S_EC_1493_1512_R ACGGTTACCTTGTTACGACT 739  134 16S_EC_1268_1287_F GAGAGCAAGCGGACCTCATA 101 TRNA_ALA- CCTCCTGCGTGCAAAGC 780 RRNH_EC_30_46.2_R  135 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_R ACAACACGAGCTGACGAC 719  137 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_ ACAACACGAGCTGICGAC 721 I14_R  138 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_ ACAACACGAGCIGACGAC 718 I12_R  139 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_ ACAACACGAGTTGACGAC 722 I11_R  140 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_ ACAACACGAGCTGACIAC 720 I16_R  141 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_ ACAACACGAICTIACGAC 723 2I_R  142 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_ ACAACACIAICTIACGAC 724 3I_R  143 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1061_1078.2_ ACAACACIAICTIACIAC 725 4I_R  147 23S_EC_2652_2669_F CTAGTACGAGAGGACCGG 79 23S_EC_2741_2760_R ACTTAGATGCTTTCAGCGGT 743  158 16S_EC_683_700_F GTGTAGCGGTGAAATGCG 137 16S_EC_880_894_R CGTACTCCCCAGGCG 796  159 16S_EC_1100_1116_F CAACGAGCGCAACCCTT 42 16S_EC_1174_1188_R TCCCCACCTTCCTCC 1019  215 SSPE_BA_121_137_F AACGCACAATCAGAAGC 3 SSPE_BA_197_216_R TCTGTTTCAGTTGCAAATTC 1132  220 GROL_EC_941_959_F TGGAAGATCTGGGTCAGGC 544 GROL_EC_1039_1060_R CAATCTGCTGACGGATCTGAGC 759  221 INFB_EC_1103_1124_F GTCGTGAAAACGAGCTGGAAGA 133 INFB_EC_1174_1191_R CATGATGGTCACAACCGG 764  222 HFLB_EC_1082_1102_F TGGCGAACCTGGTGAACGAAGC 569 EFLB_EC_1144_1168_R CTTTCGCTTTCTCGAACTCAA 802 CCAT  223 INFB_EC_1969_1994_F CGTCAGGGTAAATTCCGTGAAG 74 INFB_EC_2038_2058_R AACTTCGCCTTCGGTCATGTT 713 TTAA  224 GROL_EC_219_242_F GGTGAAAGAAGTTGCCTCTAAA 128 GROL_EC_328_350_R TTCAGGTCCATCGGGTTCATG 1377 GC CC  225 VALS_EC_1105_1124_F CGTGGCGGCGTGGTTATCGA 77 VALS_EC_1195_1214_R ACGAACTGGATGTCGCCGTT 732  226 16S_EC_556_575_F CGGAATTACTGGGCGTAAAG 70 16S_EC_683_700_R CGCATTTCACCGCTACAC 791  227 RPOC_EC_1256_1277_F ACCCAGTGCTGCTGAACCGTGC 16 RPOC_EC_1295_1315_R GTTCAAATGCCTGGATACCCA 843  228 16S_EC_774_795_F GGGAGCAAACAGGATTAGATAC 122 16S_EC_880_894_R CGTACTCCCCAGGCG 796  229 RPOC_EC_1584_1604_F TGGCCCGAAAGAAGCTGAGCG 567 RPOC_EC_1623_1643_R ACGCGGGCATGCAGAGATGCC 737  230 16S_EC_1082_1100_F ATGTTGGGTTAAGTCCCGC 37 16S_EC_1177_1196_R TGACGTCATCCCCACCTTCC 1158  231 16S_EC_1389_1407_F CTTGTACACACCGCCCGTC 88 16S_EC_1525_1541_R AAGGAGGTGATCCAGCC 714  232 16S_EC_1303_1323_F CGGATTGGAGTCTGCAACTCG 71 16S_EC_1389_1407_R GACGGGCGGTGTGTACAAG 808  233 23S_EC_23_37_F GGTGGATGCCTTGGC 129 23S_EC_115_130_R GGGTTTCCCCATTCGG 833  234 23S_EC_187_207_F GGGAACTGAAACATCTAAGTA 121 23S_EC_242_256_R TTCGCTCGCCGCTAC 1385  235 23S_EC_1602_1620_F TACCCCAAACCGACACAGG 184 23S_EC_1686_1703_R CCTTCTCCCGAAGTTACG 782  236 23S_EC_1685_1703_F CCGTAACTTCGGGAGAAGG 58 23S_EC_1828_1842_R CACCGGGCAGGCGTC 760  237 23S_EC_1827_1843_F GACGCCTGCCCGGTGC 99 23S_EC_1929_1949_R CCGACAAGGAATTTCGCTACC 775  238 23S_EC_2434_2456_F AAGGTACTCCGGGGATAACAGG 9 23S_EC_2490_2511_R AGCCGACATCGAGGTGCCAAAC 746 C  239 23S_EC_2599_2616_F GACAGTTCGGTCCCTATC 96 23S_EC_2653_2669_R CCGGTCCTCTCGTACTA 777  240 23S_EC_2653_2669_F TAGTACGAGAGGACCGG 227 23S_EC_2737_2758_R TTAGATGCTTTCAGCACTTATC 1369  241 23S_BS_−68_−44_F AAACTAGATAACAGTAGACATC 1 23S_BS_5_21_R GTGCGCCCTTTCTAACTT 841 AC  242 16S_EC_8_27_F AGAGTTTGATCATGGCTCAG 23 16S_EC_342_358_R ACTGCTGCCTCCCGTAG 742  243 16S_EC_314_332_F CACTGGAACTGAGACACGG 48 16S_EC_556_575_R CTTTACGCCCAGTAATTCCG 801  244 16S_EC_518 536_F CCAGCAGCCGCGGTAATAC 57 16S_EC_774_795_R GTATCTAATCCTGTTTGCTCCC 839  245 16S_EC_683_700_F GTGTAGCGGTGAAATGCG 137 16S_EC_967_985_R GGTAAGGTTCTTCGCGTTG 835  246 16S_EC_937_954_F AAGCGGTGGAGCATGTGG 7 16S_EC_1220_1240_R ATTGTAGCACGTGTGTAGCCC 757  247 16S_EC_1195_1213_F CAAGTCATCATGGCCCTTA 46 16S_EC_1525_1541_R AAGGAGGTGATCCAGCC 714  248 16S_EC_8_27_F AGAGTTTGATCATGGCTCAG 23 16S_EC_1525_1541_R AAGGAGGTGATCCAGCC 714  249 23S_EC_1831_1849_F ACCTGCCCAGTGCTGGAAG 18 23S_EC_1919_1936_R TCGCTACCTTAGGACCGT 1080  250 16S_EC_1387_1407_F GCCTTGTACACACCTCCCGTC 112 16S_EC_1494_1513_R CACGGCTACCTTGTTACGAC 761  251 16S_EC_1390_1411_F TTGTACACACCGCCCGTCATAC 693 16S_EC_1486_1505_R CCTTGTTACGACTTCACCCC 783  252 16S_EC_1367_1387_F TACGGTGAATACGTTCCCGGG 191 16S_EC_1485_1506_R ACCTTGTTACGACTTCACCCCA 731  253 16S_EC_804_822_F ACCACGCCGTAAACGATGA 14 16S_EC_909_929_R CCCCCGTCAATTCCTTTGAGT 773  254 16S_EC_791_812_F GATACCCTGGTAGTCCACACCG 106 16S_EC_886_904_R GCCTTGCGACCGTACTCCC 817  255 16S_EC_789_810_F TAGATACCCTGGTAGTCCACGC 206 16S_EC_882_899_R GCGACCGTACTCCCCAGG 818  256 16S_EC_1092_1109_F TAGTCCCGCAACGAGCGC 228 16S_EC_1174_1195_R GACGTCATCCCCACCTTCCTCC 810  257 23S_EC_2586_2607_F TAGAACGTCGCGAGACAGTTCG 203 23S_EC_2658_2677_R AGTCCATCCCGGTCCTCTCG 749  258 RNASEP_SA_31_49_F GAGGAAAGTCCATGCTCAC 103 RNASEP_SA_358_379_R ATAAGCCATGTTCTGTTCCATC 750  258 RNASEP_SA_31_49_F GAGGAAAGTCCATGCTCAC 103 RNASEP_EC_345_362_R ATAAGCCGGGTTCTGTCG 751  258 RNASEP_SA_31_49_F GAGGAAAGTCCATGCTCAC 103 RNASEP_BS_363_384_R GTAAGCCATGTTTTGTTCCATC 838  258 RNASEP_BS_43_61_F GAGGAAAGTCCATGCTCGC 104 RNASEP_SA_358_379_R ATAAGCCATGTTCTGTTCCATC 750  258 RNASEP_BS_43_61_F GAGGAAAGTCCATGCTCGC 104 RNASEP_EC_345_362_R ATAAGCCGGGTTCTGTCG 751  258 RNASEP_BS_43_61_F GAGGAAAGTCCATGCTCGC 104 RNASEP_BS_363_384_R GTAAGCCATGTTTTGTTCCATC 838  258 RNASEP_EC_61_77_F GAGGAAAGTCCGGGCTC 105 RNASEP_SA_358_379_R ATAAGCCATGTTCTGTTCCATC 750  258 RNASEP_EC_61_77_F GAGGAAAGTCCGGGCTC 105 RNASEP_EC_345_362_R ATAAGCCGGGTTCTGTCG 751  258 RNASEP_EC_61_77_F GAGGAAAGTCCGGGCTC 105 RNASEP_BS_363_384_R GTAAGCCATGTTTTGTTCCATC 838  259 RNASEP_BS_43_61_F GAGGAAAGTCCATGCTCGC 104 RNASEP_BS_363_384_R GTAAGCCATGTTTTGTTCCATC 838  260 RNASEP_EC_61_77_F GAGGAAAGTCCGGGCTC 105 RNASEP_EC_345_362_R ATAAGCCGGGTTCTGTCG 751  262 RNASEP_SA_31_49_F GAGGAAAGTCCATGCTCAC 103 RNASEP_SA_358_379_R ATAAGCCATGTTCTGTTCCATC 750  263 16S_EC_1082_1100_F ATGTTGGGTTAAGTCCCGC 37 16S_EC_1525_1541_R AAGGAGGTGATCCAGCC 714  264 16S_EC_556_575_F CGGAATTACTGGGCGTAAAG 70 16S_EC_774_795_R GTATCTAATCCTGTTTGCTCCC 839  265 16S_EC_1082_1100_F ATGTTGGGTTAAGTCCCGC 37 16S_EC_1177_1196_10G_ TGACGTCATGCCCACCTTCC 1160 R  266 16S_EC_1082_1100_F ATGTTGGGTTAAGTCCCGC 37 16S_EC_1177_1196_10G_ TGACGTCATGGCCACCTTCC 1161 11G_R  268 YAED_EC_513_532_F_ GGTGTTAAATAGCCTGGCAG 130 TRNA_ALA- AGACCTCCTGCGTGCAAAGC 744 MOD RRNH_EC_30_49_F_MOD  269 16S_EC_1082_1100_F_ ATGTTGGGTTAAGTCCCGC 37 16S_EC_1177_1196_R_ TGACGTCATCCCCACCTTCC 1158 MOD MOD  270 23S_EC_2586_2607_F_ TAGAACGTCGCGAGACAGTTCG 203 23S_EC_2658_2677_R_ AGTCCATCCCGGTCCTCTCG 749 MOD MOD  272 16S_EC_969_985_F ACGCGAAGAACCTTACC 19 16S_EC_1389_1407_R GACGGGCGGTGTGTACAAG 807  273 16S_EC_683_700_F GTGTAGCGGTGAAATGCG 137 16S_EC_1303_1323_R CGAGTTGCAGACTGCGATCCG 788  274 16S_EC_49_68_F TAACACATGCAAGTCGAACG 152 16S_EC_880_894_R CGTACTCCCCAGGCG 796  275 16S_EC_49_68_F TAACACATGCAAGTCGAACG 152 16S_EC_1061_1078_R ACGACACGAGCTGACGAC 734  277 CYA_BA_1349_1370_F ACAACGAAGTACAATACAAGAC 12 CYA_BA_1426_1447_R CTTCTACATTTTTAGCCATCAC 800  278 16S_EC_1090_1111_2_ TTAAGTCCCGCAACGAGCGCAA 650 16S_EC_1175_1196_R TGACGTCATCCCCACCTTCCTC 1159 F  279 16S_EC_405_432_F TGAGTGATGAAGGCCTTAGGGT 464 16S_EC_507_527_R CGGCTGCTGGCACGAAGTTAG 793 TGTAAA  280 GROL_EC_496_518_F ATGGACAAGGTTGGCAAGGAAG 34 GROL_EC_577_596_R TAGCCGCGGTCGAATTGCAT 914 G  281 GROL_EC_511_536_F AAGGAAGGCGTGATCACCGTTG 8 GROL_EC_571_593_R CCGCGGTCGAATTGCATGCCT 776 AAGA TC  288 RPOB_EC_3802_3821_F CAGCGTTTCGGCGAAATGGA 51 RPOB_EC_3862_3885_R CGACTTGACGGTTAACATTTC 786 CTG  289 RPOB_EC_3799_3821_F GGGCAGCGTTTCGGCGAAATGG 124 RPOB_EC_3862_3888_R GTCCGACTTGACGGTCAACAT 840 A TTCCTG  290 RPOC_EC_2146_2174_F CAGGAGTCGTTCAACTCGATCT 52 RPOC_EC_2227_2245_R ACGCCATCAGGCCACGCAT 736 ACATGAT  291 ASPS_EC_405_422_F GCACAACCTGCGGCTGCG 110 ASPS_EC_521_538_R ACGGCACGAGGTAGTCGC 738  292 RPOC_EC_1374_1393_F CGCCGACTTCGACGGTGACC 69 RPOC_EC_1437_1455_R GAGCATCAGCGTGCGTGCT 811  293 TUFB_EC_957_979_F CCACACGCCGTTCTTCAACAAC 55 TUFB_EC_1034_1058_R GGCATCACCATTTCCTTGTCC 829 T TTCG  294 16S_EC_7_33_F GAGAGTTTGATCCTGGCTCAGA 102 16S_EC_101_122_R TGTTACTCACCCGTCTGCCAC 1345 ACGAA T  295 VALS_EC_610_649_F ACCGAGCAAGGAGACCAGC 17 VALS_EC_705_727_R TATAACGCACATCGTCAGGGT 929 GA  344 16S_EC_971_990_F GCGAAGAACCTTACCAGGTC 113 16S_EC_1043_1062_R ACAACCATGCACCACCTGTC 726  346 16S_EC_713_732_ TAGAACACCGATGGCGAAGGC 202 16S_EC_789_809_TMOD_R TCGTGGACTACCAGGGTATCTA 1110 TMOD_F  347 16S_EC_785_806_ TGGATTAGAGACCCTGGTAGTC 560 16S_EC_880_897_TMOD_R TGGCCGTACTCCCCAGGCG 1278 TMOD_F C  348 16S_EC_960_981_ TTTCGATGCAACGCGAAGAACC 706 16S_EC_1054_1073_ TACGAGCTGACGACAGCCATG 895 TMOD_F T TMOD_R  349 23S_EC_1826_1843_ TCTGACACCTGCCCGGTGC 401 23S_EC_1906_1924_ TGACCGTTATAGTTACGGCC 1156 TMOD_F TMOD_R  350 CAPC_BA_274_303_ TGATTATTGTTATCCTGTTATG 476 CAPC_BA_349_376_TMOD_ TGTAACCCTTGTCTTTGAATT 1314 TMOD_F CCATTTGAG R GTATTTGC  351 CYA_BA_1353_1379_ TCGAAGTACAATACAAGACAAA 355 CYA_BA_1448_1467_ TTGTTAACGGCTTCAAGACCC 1423 TMOD_F AGAAGG TMOD_R  352 INFB_EC_1365_1393_ TTGCTCGTGGTGCACAAGTAAC 687 INFB_EC_1439_1467_ TTGCTGCTTTCGCATGGTTAA 1411 TMOD_F GGATATTA TMOD_R TTGCTTCAA  353 LEF_BA_756_781_ TAGCTTTTGCATATTATATCGA 220 LEF_BA_843_872_ TTCTTCCAAGGATAGATTTAT 1394 TMOD_F GCCAC TMOD_R TTCTTGTTCG  354 RPOC_EC_2218_2241_ TCTGGCAGGTATGCGTGGTCTG 405 RPOC_EC_2313_2337_ TCGCACCGTGGGTTGAGATGA 1072 TMOD_F ATG TMOD_R AGTAC  355 SSPE_BA_115_137_ TCAAGCAAACGCACAATCAGAA 255 SSPE_BA_197_222_ TTGCACGTCTGTTTCAGTTGC 1402 TMOD_F GC TMOD_R AAATTC  356 RPLB_EC_650_679_ TGACCTACAGTAAGAGGTTCTG 449 RPLB_EC_739_762_ TTCCAAGTGCTGGTTTACCCC 1380 TMOD_F TAATGAACC TMOD_R ATGG  357 RPLB_EC_688_710_ TCATCCACACGGTGGTGGTGAA 296 RPLB_EC_736_757_ TGTGCTGGTTTACCCCATGGA 1337 TMOD_F GG TMOD_R GT  358 VALS_EC_1105_1124_ TCGTGGCGGCGTGGTTATCGA 385 VALS_EC_1195_1218_ TCGGTACGAACTGGATGTCGC 1093 TMOD_F TMOD_R CGTT  359 RPOB_EC_1845_1866_ TTATCGCTCAGGCGAACTCCAA 659 RPOB_EC_1909_1929_ TGCTGGATTCGCCTTTGCTAC 1250 TMOD_F C TMOD_R G  360 23S_EC_2646_2667_ TCTGTTCTTAGTACGAGAGGAC 409 23S_EC_2745_2765_ TTTCGTGCTTAGATGCTTTCA 1434 TMOD_F C TMOD_R G  361 16S_EC_1090_1111_2_ TTTAAGTCCCGCAACGAGCGCA 697 16S_EC_1175_1196_ TTGACGTCATCCCCACCTTCC 1398 TMOD_F A TMOD_R TC  362 RPOB_EC_3799_3821_ TGGGCAGCGTTTCGGCGAAATG 581 RPOB_EC_3862_3888_ TGTCCGACTTGACGGTCAACA 1325 TMOD_F GA TMOD_R TTTCCTG  363 RPOC_EC_2146_2174_ TCAGGAGTCGTTCAACTCGATC 284 RPOC_EC_2227_2245_ TACGCCATCAGGCCACGCAT 898 TMOD_F TACATGAT TMOD_R  364 RPOC_EC_1374_1393_ TCGCCGACTTCGACGGTGACC 367 RPOC_EC_1437_1455_ TGAGCATCAGCGTGCGTGCT 1166 TMOD_F TMOD_R  367 TUFB_EC_957_979_ TCCACACGCCGTTCTTCAACAA 308 TUFB_EC_1034_1058_ TGGCATCACCATTTCCTTGTC 1276 TMOD_F CT TMOD_R CTTCG  423 SP101_SPET11_893_ TGGGCAACAGCAGCGGATTGCG 580 SP101_SPET11_988_ TCATGACAGCCAAGACCTCAC 990 921_TMOD_F ATTGCGCG 1012_TMOD_R CCACC  424 SP101_SPET11_1154_ TCAATACCGCAACAGCGGTGGC 258 SP101_SPET11_1251_ TGACCCCAACCTGGCCTTTTG 1155 1179_TMOD_F TTGGG 1277_TMOD_R TCGTTGA  425 SP101_SPET11_118_ TGCTGGTGAAAATAACCCAGAT 528 SP101_SPET11_213_238_ TTGTGGCCGATTTCACCACCT 1422 147_TMOD_F GTCGTCTTC TMOD_R GCTCCT  426 SP101_SPET11_1314_ TCGCAAAAAAATCCAGCTATTA 363 SP101_SPET11_1403_ TAAACTATTTTTTTAGCTATA 849 1336_TMOD_F GC 1431_TMOD_R CTCGAACAC  427 SP101_SPET11_1408_ TCGAGTATAGCTAAAAAAATAG 359 SP101_SPET11_1486_ TGGATAATTGGTCGTAACAAG 1268 1437_TMOD_F TTTATGACA 1515_TMOD_R GGATAGTGAG  428 SP101_SPET11_1688_ TCCTATATTAATCGTTTACAGA 334 SP101_SPET11_1783_ TATATGATTATCATTGAACTG 932 1716_TMOD_F AACTGGCT 1808_TMOD_R CGGCCG  429 SP101_SPET11_1711_ TCTGGCTAAAACTTTGGCAACG 406 SP101_SPET11_1808_ TGCGTGACGACCTTCTTGAAT 1239 1733_TMOD_F GT 1835_TMOD_R TGTAATCA  430 SP101_SPET11_1807_ TATGATTACAATTCAAGAAGGT 235 SP101_SPET11_1901_ TTTGGACCTGTAATCAGCTGA 1439 1835_TMOD_F CGTCACGC 1927_TMOD_R ATACTGG  431 SP101_SPET11_1967_ TTAACGGTTATCATGGCCCAGA 649 SP101_SPET11_2062_ TATTGCCCAGAAATCAAATCA 940 1991_TMOD_F TGGG 2083_TMOD_R TC  432 SP101_SPET11_216_ TAGCAGGTGGTGAAATCGGCCA 210 SP101_SPET11_308_333_ TTGCCACTTTGACAACTCCTG 1404 243_TMOD_F CATGATT TMOD_R TTGCTG  433 SP101_SPET11_2260_ TCAGAGACCGTTTTATCCTATC 272 SP101_SPET11_2375_ TTCTGGGTGACCTGGTGTTTT 1393 2283_TMOD_F AGC 2397_TMOD_R AGA  434 SP101_SPET11_2375_ TTCTAAAACACCAGGTCACCCA 675 SP101_SPET11_2470_ TAGCTGCTAGATGAGCTTCTG 918 2399_TMOD_F GAAG 2497_TMOD_R CCATGGCC  435 SP101_SPET11_2468_ TATGGCCATGGCAGAAGCTCA 238 SP101_SPET11_2543_ TCCATAAGGTCACCGTCACCA 1007 2487_TMOD_F 2570_TMOD_R TTCAAAGC  436 SP101_SPET11_266_ TCTTGTACTTGTGGCTCACACG 417 SP101_SPET11_355_380_ TGCTGCTTTGATGGCTGAATC 1249 295_TMOD_F GCTGTTTGG TMOD_R CCCTTC  437 SP101_SPET11_2961_ TACCATGACAGAAGGCATTTTG 183 SP101_SPET11_3023_ TGGAATTTACCAGCGATAGAC 1264 2984_TMOD_F ACA 3045_TMOD_R ACC  438 SP101_SPET11_3075_ TGATGACTTTTTAGCTAATGGT 473 SP101_SPET11_3168_ TAATCGACGACCATCTTGGAA 875 3103_TMOD_F CAGGCAGC 3196_TMOD_R AGATTTCTC  439 SP101_SPET11_322_ TGTCAAAGTGGCACGTTTACTG 631 SP101_SPET11_423_441_ TATCCCCTGCTTCTGCTGCC 934 344_TMOD_F GC TMOD_R  440 SP101_SPET11_3386_ TAGCGTAAAGGTGAACCTT 215 SP101_SPET11_3480_ TCCAGCAGTTACTGTCCCCTC 1005 3403_TMOD_F 3506_TMOD_R ATCTTTG  441 SP101_SPET11_3511_ TGCTTCAGGAATCAATGATGGA 531 SP101_SPET11_3605_ TGGGTCTACACCTGCACTTGC 1294 3535_TMOD_F GCAG 3629_TMOD_R ATAAC  442 SP101_SPET11_358_ TGGGGATTCAGCCATCAAAGCA 588 SP101_SPET11_448_473_ TCCAACCTTTTCCACAACAGA 998 387_TMOD_F GCTATTGAC TMOD_R ATCAGC  443 SP101_SPET11_600_ TCCTTACTTCGAACTATGAATC 348 SP101_SPET11_686_714_ TCCCATTTTTTCACGCATGCT 1018 629_TMOD_F TTTTGGAAG TMOD_R GAAAATATC  444 SP101_SPET11_658_ TGGGGATTGATATCACCGATAA 589 SP101_SPET11_756_784_ TGATTGGCGATAAAGTGATAT 1189 684_TMOD_F GAAGAA TMOD_R TTTCTAAAA  445 SP101_SPET11_776_ TTCGCCAATCAAAACTAAGGGA 673 SP101_SPET11_871_896_ TGCCCACCAGAAAGACTAGCA 1217 801_TMOD_F ATGGC TMOD_R GGATAA  446 SP101_SPET11_1_29_ TAACCTTAATTGGAAAGAAACC 154 SP101_SPET11_92_116_ TCCTACCCAACGTTCACCAAG 1044 TMOD_F CAAGAAGT TMOD_R GGCAG  447 SP101_SPET11_364_ TCAGCCATCAAAGCAGCTATTG 276 SP101_SPET11_448_471_ TACCTTTTCCACAACAGAATC 894 385_F R AGC  448 SP101_SPET11_3085_ TAGCTAATGGTCAGGCAGCC 216 SP101_SPET11_3170_ TCGACGACCATCTTGGAAAGA 1066 3104_F 3194_R TTTC  449 RPLB_EC_690_710_F TGTGCTGGTTTACCCCATGGA 309 RPLB_EC_737_758_R TGTGCTGGTTTACCCCATGGAG 1336  481 BONTA_X52066_538_ TATGGCTCTACTCAA 239 BONTA_X52066_647_660_ TGTTACTGCTGGAT 1346 552_F R  482 BONTA_X52066_538_ TA*TpGGC*Tp*Cp*TpA*Cp* 143 BONTA_X52066_647_ TG*Tp*TpA*Cp*TpG*Cp*T 1146 552P_F Tp*CpAA 660P_R pGGAT  483 BONTA_X52066_701_ GAATAGCAATTAATCCAAAT 94 BONTA_X52066_759_775_ TTACTTCTAACCCACTC 1367 720_F R  484 BONTA_X52066_701_ GAA*TpAG*CpAA*Tp*TpAA* 91 BONTA_X52066_759_ TTA*Cp*Tp*Tp*Cp*TpAA* 1359 720P_F Tp*Cp*CpAAAT 775P_R Cp*Cp*CpA*Cp*TpC  485 BONTA_X52066_450_ TCTAGTAATAATAGGACCCTCA 393 BONTA_X52066_517_539_ TAACCATTTCGCGTAAGATTC 859 473_F GC R AA  486 BONTA_X52066_450_ T*Cp*TpAGTAATAATAGGA*C 142 BONTA_X52066_517_ TAACCA*Tp*Tp*Tp*CpGCG 857 473P_F p*Cp*Cp*Tp*CpAGC 539P_R TAAGA*Tp*Tp*CpAA  487 BONTA_X52066_591_ TGAGTCACTTGAAGTTGATACA 463 BONTA_X52066_644_671_ TCATGTGCTAATGTTACTGCT 992 620_F AATCCTCT R GGATCTG  608 SSPE_BA_156_168P_F TGGTpGCpTpAGCpATT 616 SSPE_BA_243_255P_R TGCpAGCpTGATpTpGT 1241  609 SSPE_BA_75_89P_F TACpAGAGTpTpTpGCpGAC 192 SSPE_BA_163_177P_R TGTGCTpTpTpGAATpGCpT 1338  610 SSPE_BA_150_168P_F TGCTTCTGGTpGCpTpAGCpAT 533 SSPE_BA_243_264P_4 TGATTGTTTTGCpAGCpTGAT 1191 T  611 SSPE_BA_72_89P_F TGGTACpAGAGTpTpTpGCpGA 602 SSPE_BA_163_182P_R TCATTTGTGCTpTpTpGAATp 995 C GCpT  612 SSPE_BA_114_137P_F TCAAGCAAACGCACAATpCpAG 255 SSPE_BA_196_222P_R TTGCACGTCpTpGTTTCAGTT 1401 AAGC GCAAATTC  699 SSPE_BA_123_153_F TGCACAATCAGAAGCTAAGAAA 488 SSPE_BA_202_231_R TTTCACAGCATGCACGTCTGT 1431 GCGCAAGCT TTCAGTTGC  700 SSPE_BA_156_168_F TGGTGCTAGCATT 612 SSPE_BA_243_255_R TGCAGCTGATTGT 1202  701 SSPE_BA_75_89_F TACAGAGTTTGCGAC 179 SSPE_BA_163_177_R TGTGCTTTGAATGCT 1338  702 SSPE_BA_150_168_F TGCTTCTGGTGCTAGCATT 533 SSPE_BA_243_264_R TGATTGTTTTGCAGCTGATTG 1190 T  703 SSPE_BA_72_89_F TGGTACAGAGTTTGCGAC 600 SSPE_BA_163_182_R TCATTTGTGCTTTGAATGCT 995  704 SSPE_BA_146_168_F TGCAAGCTTCTGGTGCTAGCAT 484 SSPE_BA_242_267_R TTGTGATTGTTTTGCAGCTGA 1421 T TTGTG  705 SSPE_BA_63_89_F TGCTAGTTATGGTACAGAGTTT 518 SSPE_BA_163_191_R TCATAACTAGCATTTGTGCTT 986 GCGAC TGAATGCT  706 SSPE_BA_114_137_F TCAAGCAAACGCACAATCAGAA 255 SSPE_BA_196_222_R TTGCACGTCTGTTTCAGTTGC 1402 GC AAATTC  770 PLA_AF053945_7377_ TGACATCCGGCTCACGTTATTA 442 PLA_AF053945_7434_ TGTAAATTCCGCAAAGACTTT 1313 7402_F TGGT 7462_R GGCATTAG  771 PLA_AF053945_7382_ TCCGGCTCACGTTATTATGGTA 327 PLA_AF053945_7482_ TGGTCTGAGTACCTCCTTTGC 1304 7404_F C 7502_R  772 PLA_AF053945_7481_ TGCAAAGGAGGTACTCAGACCA 481 PLA_AF053945_7539_ TATTGGAAATACCGGCAGCAT 943 7503_F T 7562_R CTC  773 PLA_AF053945_7186_ TTATACCGGAAACTTCCCGAAA 657 PLA_AF053945_7257_ TAATGCGATACTGGCCTGCAA 879 7211_F GGAG 7280_R GTC  774 CAF1_AF053947_ TCAGTTCCGTTATCGCCATTGC 292 CAF1_AF053947_33494_ TGCGGGCTGGTTCAACAAGAG 1235 33407_33430_F AT 33514_R  775 CAF1_AF053947_ TCACTCTTACATATAAGGAAGG 270 CAF1_AF053947_33595_ TCCTGTTTTATAGCCGCCAAG 1053 33515_33541_F CGCTC 33621_R AGTAAG  776 CAF1_AF053947_ TGGAACTATTGCAACTGCTAAT 542 CAF1_AF053947_33499_ TGATGCGGGCTGGTTCAAC 1183 33435_33457_F G 33517_R  777 CAF1_AF053947_ TCAGGATGGAAATAACCACCAA 286 CAF1_AF053947_33755_ TCAAGGTTCTCACCGTTTACC 962 33687_33716_F TTCACTAC 33782_R TTAGGAG  778 INV_U22457_515_539_ TGGCTCCTTGGTATGACTCTGC 573 INV_U22457_571_598_R TGTTAAGTGTGTTGCGGCTGT 1343 F TTC CTTTATT  779 INV_U22457_699_724_ TGCTGAGGCCTGGACCGATTAT 525 INV_U22457_753_776_R TCACGCGACGAGTGCCATCCA 976 F TTAC TTG  780 INV_U22457_834_858_ TTATTTACCTGCACTCCCACAA 664 INV_U22457_942_966_R TGACCCAAAGCTGAAAGCTTT 1154 F CTG ACTG  781 INV_U22457_1558_ TGGTAACAGAGCCTTATAGGCG 597 INV_U22457_1619_1643_ TTGCGTTGCAGATTATCTTTA 1408 1581_F CA R CCAA  782 LL_NC003143_ TGTAGCCGCTAAGCACTACCAT 627 LL_NC003143_2367073_ TCTCATCCCGATATTACCGCC 1123 2366996_2367019_F CC 2367097_R ATGA  783 LL_NC003143_ TGGACGGCATCACGATTCTCTA 550 LL_NC003143_2367249_ TGGCAACAGCTCAACACCTTT 1272 2367172_2367194_F C 2367271_R GG  874 RPLB_EC_649_679_F TGICCIACIGTIIGIGGTTCTG 620 RPLB_EC_739_762_TMOD_ TTCCAAGTGCTGGTTTACCCC 1380 TAATGAACC R ATGG  875 RPLB_EC_642_679P_F TpCpCpTpTpGITpGICCIACI 646 RPLB_EC_739_762_TMOD_ TTCCAAGTGCTGGTTTACCCC 1380 GTTIGIGGTTCTGTAATGAACC R ATGG  876 MECIA_Y14051_3315_ TTACACATATCGTGAGCAATGA 653 MECIA_Y14051_3367_ TGTGATATGGAGGTGTAGAAG 1333 3341_F ACTGA 3393_R GTGTTA  877 MECA_Y14051_3774_ TAAAACAAACTACGGTAACATT 144 MECA_Y14051_3828_ TCCCAATCTAACTTCCACATA 1015 3802_F GATCGCA 3854_R CCATCT  878 MECA_Y14051_3645_ TGAAGTAGAAATGACTGAACGT 434 MECA_Y14051_3690_ TGATCCTGAATGTTTATATCT 1181 3670_F CCGA 3719_R TTAACGCCT  879 MECA_Y14051_4507_ TCAGGTACTGCTATCCACCCTC 288 MECA_Y14051_4555_ TGGATAGACGTCATATGAAGG 1269 4530_F AA 4581_R TGTGCT  880 MECA_Y14051_4510_ TGTACTGCTATCCACCCTCAA 626 MECA_Y14051_4586_ TATTCTTCGTTACTCATGCCA 939 4530_F 4610_R TACA  881 MECA_Y14051_4669_ TCACCAGGTTCAACTCAAAAAA 262 MECA_Y14051_4765_ TAACCACCCCAAGATTTATCT 858 4698_F TATTAACA 4793_R TTTTGCCA  882 MECA_Y14051_4520_ TCpCpACpCpCpTpCpAA 389 MECA_Y14051_4590_ TpACpTpCpATpGCpCpA 1357 4530P_F 4600P_R  883 MECA_Y14051_4520_ TCpCpACpCpCpTpCpAA 389 MECA_Y14051_4600_ TpATpTpCpTpTpCpGTpT 1358 4530P_F 4610P_R  902 TRPE_AY094355_1467_ ATGTCGATTGCAATCCGTACTT 36 TRPE_AY094355_1569_ TGCGCGAGCTTTTATTTGGGT 1231 1491_F GTG 1592_R TTC  903 TRPE_AY094355_1445_ TGGATGGCATGGTGAAATGGAT 557 TRPE_AY094355_1551_ TATTTGGGTTTCATTCCACTC 944 1471_F ATGTC 1580_R AGATTCTGG  904 TRPE_AY094355_1278_ TCAAATGTACAAGGTGAAGTGC 247 TRPE_AY094355_1392_ TCCTCTTTTCACAGGCTCTAC 1048 1303_F GTGA 1418_R TTCATC  905 TRPE AY094355_1064_ TCGACCTTTGGCAGGAACTAGA 357 TRPE_AY094355_1171_ TACATCGTTTCGCCCAAGATC 885 1086_F C 1196_R AATCA  906 TRPE_AY094355_666_ GTGCATGCGGATACAGAGCAGA 135 TRPE_AY094355_769_ TTCAAAATGCGGAGGCGTATG 1372 688_F G 791_R TG  907 TRPE_AY094355_757_ TGCAAGCGCGACCACATACG 483 TRPE_AY094355_864_ TGCCCAGGTACAACCTGCAT 1218 776_F 883_R  908 RECA_AF251469_43_ TGGTACATGTGCCTTCATTGAT 601 RECA_AF251469_140_ TTCAAGTGCTTGCTCACCATT 1375 68_F GCTG 163_R GTC  909 RECA_AF251469_169_ TGACATGCTTGTCCGTTCAGGC 446 RECA_AF251469_277_ TGGCTCATAAGACGCGCTTGT 1280 190_F 300_R AGA  910 PARC_X95819_87_110_ TGGTGACTCGGCATGTTATGAA 609 PARC_X95819_201_222_ TTCGGTATAACGCATCGCAGC 1387 F GC R A  911 PARC_X95819_87_110_ TGGTGACTCGGCATGTTATGAA 609 PARC_X95819_192_219_ GGTATAACGCATCGCAGCAAA 836 F GC R AGATTTA  912 PARC_X95819_123_ GGCTCAGCCATTTAGTTACCGC 120 PARC_X95819_232_260_ TCGCTCAGCAATAATTCACTA 1081 147_F TAT R TAAGCCGA  913 PARC_X95819_43_63_F TCAGCGCGTACAGTGGGTGAT 277 PARC_X95819_143_170_ TTCCCCTGACCTTCGATTAAA 1383 R GGATAGC  914 OMPA_AY485227_272_ TTACTCCATTATTGCTTGGTTA 655 OMPA_AY485227_364_ GAGCTGCGCCAACGAATAAAT 812 301_F CACTTTCC 388_R CGTC  915 OMPA_AY485227_379_ TGCGCAGCTCTTGGTATCGAGT 509 OMPA_AY485227_492_ TGCCGTAACATAGAAGTTACC 1223 401_F T 519_R GTTGATT  916 OMPA_AY485227_311_ TACACAACAATGGCGGTAAAGA 178 OMPA_AY485227_424_ TACGTCGCCTTTAACTTGGTT 901 335_F TGG 453_R ATATTCAGC  917 OMPA_AY485227_415_ TGCCTCGAAGCTGAATATAACC 506 OMPA_AY485227_514_ TCGGGCGTAGTTTTTAGTAAT 1092 441_F AAGTT 546_R TAAATCAGAAGT  918 OMPA_AY485227_494_ TCAACGGTAACTTCTATGTTAC 252 OMPA_AY485227_569_ TCGTCGTATTTATAGTGACCA 1108 520_F TTCTG 596_R GCACCTA  919 OMPA_AY485227_551_ TCAAGCCGTACGTATTATTAGG 257 OMPA_AY485227_658_ TTTAAGCGCCAGAAAGCACCA 1425 577_F TGCTG 680_R AC  920 OMPA_AY485227_555_ TCCGTACGTATTATTAGGTGCT 328 OMPA_AY485227_635_ TCAACACCAGCGTTACCTAAA 954 581_F GGTCA 662_R GTACCTT  921 OMPA_AY485227_556_ TCGTACGTATTATTAGGTGCTG 379 OMPA_AY485227_659_ TCGTTTAAGCGCCAGAAAGCA 1114 583_F GTCACT 683_R CCAA  922 OMPA_AY485227_657_ TGTTGGTGCTTTCTGGCGCTTA 645 OMPA_AY485227_739_ TAAGCCAGCAAGAGCTGTATA 871 679_F A 765_R GTTCCA  923 OMPA_AY485227_660_ TGGTGCTTTCTGGCGCTTAAAC 613 OMPA_AY485227_786_ TACAGGAGCAGCAGGCTTCAA 884 683_F GA 807_R G  924 GYRA_AF100557_4_23_ TCTGCCCGTGTCGTTGGTGA 402 GYRA_AF100557_119_ TCGAACCGAAGTTACCCTGAC 1063 F 142_R CAT  925 GYRA_AF100557_70_ TCCATTGTTCGTATGGCTCAAG 316 GYRA_AF100557_178_ TGCCAGCTTAGTCATACGGAC 1211 94_F ACT 201_R TTC  926 GYRB_AB008700_19_ TCAGGTGGCTTACACGGCGTAG 289 GYRB_AB008700_111_ TATTGCGGATCACCATGATGA 941 40_F 140_R TATTCTTGC  927 GYRB_AB008700_265_ TCTTTCTTGAATGCTGGTGTAC 420 GYRB_AB008700_369_ TCGTTGAGATGGTTTTTACCT 1113 292_F GTATCG 395_R TCGTTG  928 GYRB_AB008700_368_ TCAACGAAGGTAAAAACCATCT 251 GYRB_AB008700_466_ TTTGTGAAACAGCGAACATTT 1440 394_F CAACG 494_R TCTTGGTA  929 GYRB_AB008700_477_ TGTTCGCTGTTTCACAAACAAC 641 GYRB_AB008700_611_ TCACGCGCATCATCACCAGTC 977 504_F ATTCCA 632_R A  930 GYRB_AB008700_760_ TACTTACTTGAGAATCCACAAG 198 GYRB_AB008700_862_ ACCTGCAATATCTAATGCACT 729 787_F CTGCAA 888_R CTTACG  931 WAAA_Z96925_2_29_F TCTTGCTCTTTCGTGAGTTCAG 416 WAAA_Z96925_115_138_R CAAGCGGTTTGCCTCAAATAG 758 TAAATG TCA  932 WAAA_Z96925_286_ TCGATCTGGTTTCATGCTGTTT 360 WAAA_Z96925_394_412_ TGGCACGAGCCTGACCTGT 1274 311_F CAGT R  939 RPOB_EC_3798_3821_F TGGGCAGCGTTTCGGCGAAATG 581 RPOB_EC_3862_3889_R TGTCCGACTTGACGGTCAGCA 1326 GA TTTCCTG  940 RPOB_EC_3798_3821_F TGGGCAGCGTTTCGGCGAAATG 581 RPOB_EC_3862_3889_2_ TGTCCGACTTGACGGTTAGCA 1327 GA R TTTCCTG  941 TUFB_EC_275_299_F TGATCACTGGTGCTGCTCAGAT 468 TUFB_EC_337_362_R TGGATGTGCTCACGAGTCTGT 1271 GGA GGCAT  942 TUFB_EC_251_278_F TGCACGCCGACTATGTTAAGAA 493 TUFB_EC_337_360_R TATGTGCTCACGAGTTTGCGG 937 CATGAT CAT  949 GYRB_AB008700_760_ TACTTACTTGAGAATCCACAAG 198 GYRB_AB008700_862_ TCCTGCAATATCTAATGCACT 1050 787_F CTGCAA 888_2_R CTTACG  958 RPOC_EC_2223_2243_F TGGTATGCGTGGTCTGATGGC 605 RPOC_EC_2329_2352_R TGCTAGACCTTTACGTGCACC 1243 GTG  959 RPOC_EC_918_938_F TCTGGATAACGGTCGTCGCGG 404 RPOC_EC_1009_1031_R TCCAGCAGGTTCTGACGGAAA 1004 CG  960 RPOC_EC_2334_2357_F TGCTCGTAAGGGTCTGGCGGAT 523 RPOC_EC_2380_2403_R TACTAGACGACGGGTCAGGTA 905 AC ACC  961 RPOC_EC_917_938_F TATTGGACAACGGTCGTCGCGG 242 RPOC_EC_1009_1034_R TTACCGAGCAGGTTCTGACGG 1362 AAACG  962 RPOB_EC_2005_2027_F TCGTTCCTGGAACACGATGACG 387 RPOB_EC_2041_2064_R TTGACGTTGCATGTTCGAGCC 1399 C CAT  963 RPOB_EC_1527_1549_F TCAGCTGTCGCAGTTCATGGAC 282 RPOB_EC_1630_1649_R TCGTCGCGGACTTCGAAGCC 1104 C  964 INFB_EC_1347_1367_F TGCGTTTACCGCAATGCGTGC 515 INFB_EC_1414_1432_R TCGGCATCACGCCGTCGTC 1090  965 VALS_EC_1128_1151_F TATGCTGACCGACCAGTGGTAC 237 VALS_EC_1231_1257_R TTCGCGCATCCAGGAGAAGTA 1384 GT CATGTT  978 RPOC_EC_2145_2175_F TCAGGAGTCGTTCAACTCGATC 285 RPOC_EC_2228_2247_R TTACGCCATCAGGCCACGCA 1363 TACATGATG 1045 CJST_CJ_1668_1700_F TGCTCGAGTGATTGACTTTGCT 522 CJST_CJ_1774_1799_R TGAGCGTGTGGAAAAGGACTT 1170 AAATTTAGAGA GGATG 1046 CJST_CJ_2171_2197_F TCGTTTGGTGGTGGTAGATGAA 388 CJST_CJ_2283_2313_R TCTCTTTCAAAGCACCATTGC 1126 AAAGG TCATTATAGT 1047 CJST_CJ_584_616_F TCCAGGACAAATGTATGAAAAA 315 CJST_CJ_663_692_R TTCATTTTCTGGTCCAAAGTA 1379 TGTCCAAGAAG AGCAGTATC 1048 CJST_CJ_360_394_F TCCTGTTATCCCTGAAGTAGTT 346 CJST_CJ_442_476_R TCAACTGGTTCAAAAACATTA 955 AATCAAGTTTGTT AGTTGTAATTGTCC 1049 CJST_CJ_2636_2668_F TGCCTAGAAGATCTTAAAAATT 504 CJST_CJ_2753_2777_R TTGCTGCCATAGCAAAGCCTA 1409 TCCGCCAACTT CAGC 1050 CJST_CJ_1290_1320_F TGGCTTATCCAAATTTAGATCG 575 CJST_CJ_1406_1433_R TTTGCTCATGATCTGCATGAA 1437 TGGTTTTAC GCATAAA 1051 CJST_CJ_3267_3293_F TTTGATTTTACGCCGTCCTCCA 707 CJST_CJ_3356_3385_R TCAAAGAACCCGCACCTAATT 951 GGTCG CATCATTTA 1052 CJST_CJ_5_39_F TAGGCGAAGATATACAAAGAGT 222 CJST_CJ_104_137_R TCCCTTATTTTTCTTTCTACT 1029 ATTAGAAGCTAGA ACCTTCGGATAAT 1053 CJST_CJ_1080_1110_F TTGAGGGTATGCACCGTCTTTT 681 CJST_CJ_1166_1198_R TCCCCTCATGTTTAAATGATC 1022 TGATTCTTT AGGATAAAAAGC 1054 CJST_CJ_2060_2090_F TCCCGGACTTAATATCAATGAA 323 CJST_CJ_2148_2174_R TCGATCCGCATCACCATCAAA 1068 AATTGTGGA AGCAAA 1055 CJST_CJ_2869_2895_F TGAAGCTTGTTCTTTAGCAGGA 432 CJST_CJ_2979_3007_R TCCTCCTTGTGCCTCAAAACG 1045 CTTCA CATTTTTA 1056 CJST_CJ_1880_1910_F TCCCAATTAATTCTGCCATTTT 317 CJST_CJ_1981_2011_R TGGTTCTTACTTGCTTTGCAT 1309 TCCAGGTAT AAACTTTCCA 1057 CJST_CJ_2185_2212_F TAGATGAAAAGGGCGAAGTGGC 208 CJST_CJ_2283_2316_R TGAATTCTTTCAAAGCACCAT 1152 TAATGG TGCTCATTATAGT 1058 CJST_CJ_1643_1670_F TTATCGTTTGTGGAGCTAGTGC 660 CJST_CJ_1724_1752_R TGCAATGTGTGCTATGTCAGC 1198 TTATGC AAAAAGAT 1059 CJST_CJ_2165_2194_F TGCGGATCGTTTGGTGGTTGTA 511 CJST_CJ_2247_2278_R TCCACACTGGATTGTAATTTA 1002 GATGAAAA CCTTGTTCTTT 1060 CJST_CJ_599_632_F TGAAAAATGTCCAAGAAGCATA 424 CJST_CJ_711_743_R TCCCGAACAATGAGTTGTATC 1024 GCAAAAAAAGCA AACTATTTTTAC 1061 CJST_CJ_360_393_F TCCTGTTATCCCTGAAGTAGTT 345 CJST_CJ_443_477_R TACAACTGGTTCAAAAACATT 882 AATCAAGTTTGT AAGCTGTAATTGTC 1062 CJST_CJ_2678_2703_F TCCCCAGGACACCCTGAAATTT 321 CJST_CJ_2760_2787_R TGTGCTTTTTTTGCTGCCATA 1339 CAAC GCAAAGC 1063 CJST_CJ_1268_1299_F AGTTATAAACACGGCTTTCCTA 29 CJST_CJ_1349_1379_R TCGGTTTAAGCTCTACATGAT 1096 TGGCTTATCC CGTAAGGATA 1064 CJST_CJ_1680_1713_F TGATTTTGCTAAATTTAGAGAA 479 CJST_CJ_1795_1822_R TATGTGTAGTTGAGCTTACTA 938 ATTGCGGATGAA CATGAGC 1065 CJST_CJ_2857_2887_F TGGCATTTCTTATGAAGCTTGT 565 CJST_CJ_2965_2998_R TGCTTCAAAACGCATTTTTAC 1253 TCTTTAGCA ATTTTCGTTAAAG 1070 RNASEP_BKM_580_599_ TGCGGGTAGGGAGCTTGAGC 512 RNASEP_BKM_665_686_R TCCGATAAGCCGGATTCTGTG 1034 F C 1071 RNASEP_BKM_616_637_ TCCTAGAGGAATGGCTGCCACG 333 RNASEP_BKM_665_687_R TGCCGATAAGCCGGATTCTGT 1222 F GC 1072 RNASEP_BDP_574_592_ TGGCACGGCCATCTCCGTG 561 RNASEP_BDP_616_635_R TCGTTTCACCCTGTCATGCCG 1115 F 1073 23S_BRM_1110_1129_F TGCGCGGAAGATGTAACGGG 510 23S_BRM 1176_1201_R TCGCAGGCTTACAGAACGCTC 1074 TCCTA 1074 23S_BRM_515_536_F TGCATACAAACAGTCGGAGCCT 496 23S_BRM_616_635_R TCGGACTCGCTTTCGCTACG 1088 1075 RNASEP_CLB_459_487_ TAAGGATAGTGCAACAGAGATA 162 RNASEP_CLB_498_526_R TGCTCTTACCTCACCGTTCCA 1247 F TACCGCC CCCTTACC 1076 RNASEP_CLB_459_487_ TAAGGATAGTGCAACAGAGATA 162 RNASEP_CLB_498_522_R TTTACCTCGCCTTTCCACCCT 1426 F TACCGCC TACC 1077 ICD_CXB_93_120_F TCCTGACCGACCCATTATTCCC 343 ICD_CXB_172_194_R TAGGATTTTTCCACGGCGGCA 921 TTTATC TC 1078 ICD_CXB_92_120_F TTCCTGACCGACCCATTATTCC 671 ICD_CXB_172_194_R TAGGATTTTTCCACGGCGGCA 921 CTTTATC TC 1079 ICD_CXB_176_198_F TCGCCGTGGAAAAATCCTACGC 369 ICD_CXB_224_247_R TAGCCTTTTCTCCGGCGTAGA 916 T TCT 1080 IS1111A_NC002971_ TCAGTATGTATCCACCGTAGCC 290 IS1111A_NC002971_ TAAACGTCCGATACCAATGGT 848 6866_6891_F AGTC 6928_6954_R TCGCTC 1081 IS1111A_NC002971_ TGGGTGACATTCATCAATTTCA 594 IS1111A_NC002971_ TCAACAACACCTCCTTATTCC 952 7456_7483_F TCGTTC 7529_7554_R CACTC 1082 RNASEP_RKP_419_448_ TGGTAAGAGCGCACCGGTAAGT 599 RNASEP_RKP_542_565_R TCAAGCGATCTACCCGCATTA 957 F TGGTAACA CAA 1083 RNASEP_RKP_422_443_ TAAGAGCGCACCGGTAAGTTGG 159 RNASEP_RKP_542_565_R TCAAGCGATCTACCCGCATTA 957 F CAA 1084 RNASEP_RKP_466_491_ TCCACCAAGAGCAAGATCAAAT 310 RNASEP_RKP_542_565_R TCAAGCGATCTACCCGCATTA 957 F AGGC CAA 1085 RNASEP_RKP_264_287_ TCTAAATGGTCGTGCAGTTGCG 391 RNASEP_RKP_295_321_R TCTATAGAGTCCGGACTTTCC 1119 F TG TCGTGA 1086 RNASEP_RKP_426_448_ TGCATACCGGTAAGTTGGCAAC 497 RNASEP_RKP_542_565_R TCAAGCGATCTACCCGCATTA 957 F A CAA 1087 OMPB_RKP_860_890_F TTACAGGAAGTTTAGGTGGTAA 654 OMPB_RKP_972_996_R TCCTGCAGCTCTACCTGCTCC 1051 TCTAAAAGG ATTA 1088 OMPB_RKP_1192_ TCTACTGATTTTGGTAATCTTG 392 OMPB_RKP_1288_1315_R TAGCAgCAAAAGTTATCACAC 910 1221_F CAGCACAG CTGCAGT 1089 OMPE_RKP_3417_ TGCAAGTGGTACTTCAACATGG 485 OMPB_RKP_3520_3550_R TGGTTGTAGTTCCTGTAGTTG 1310 3440_F GG TTGCATTAAC 1090 GLTA_RKP_1043_ TGGGACTTGAAGCTATCGCTCT 576 GLTA_RKP_1138_1162_R TGAACATTTGCGACGGTATAC 1147 1072_F TAAAGATG CCAT 1091 GLTA_RKP_400_428_F TCTTCTCATCCTATGGCTATTA 413 GLTA_RKP_499_529_R TGGTGGGTATCTTAGCAATCA 1305 TGCTTGC TTCTAATAGC 1092 GLTA_RKP_1023_ TCCGTTCTTACAAATAGCAATA 330 GLTA_RKP_1129_1156_R TTGGCGACGGTATACCCATAG 1415 1055_F GAACTTGAAGC CTTTATA 1093 GLTA_RKP_1043_ TGGAGCTTGAAGCTATCGCTCT 553 GLTA_RKP_1138_1162_R TGAACATTTGCGACGGTATAC 1147 1072_2_F TAAAGATG CCAT 1094 GLTA_RKP_1043_ TGGAACTTGAAGCTCTCGCTCT 543 GLTA_RKP_1138_1164_R TGTGAACATTTGCGACGGTAT 1330 1072_3_F TAAAGATG ACCCAT 1095 GLTA_RKP_400_428_F TCTTCTCATCCTATGGCTATTA 413 GLTA_RKP_505_534_R TGCGATGGTAGGTATCTTAGC 1230 TGCTTGC AATCATTCT 1096 CTXA_VBC_117_142_F TCTTATGCCAAGAGGACAGAGT 410 CTXA_VBC_194_218_R TGCCTAACAAATCCCGTCTGA 1226 GAGT GTTC 1097 CTXA_VEC_351_377_F TGTATTAGGGGCATACAGTCCT 630 CTXA_VBC_441_466_R TGTCATCAAGCACCCCAAAAT 1324 CATCC GAACT 1098 RNASEP_VBC_331_349_ TCCGCGGAGTTGACTGGGT 325 RNASEP_VBC_388_414_R TGACTTTCCTCCCCCTTATCA 1163 F GTCTCC 1099 TOXR_VBC_135_158_F TCGATTAGGCAGCAACGAAAGC 362 TOXR_VBC_221_246_R TTCAAAACCTTGCTCTCGCCA 1370 CG AACAA 1100 ASD_FRT_1_29_F TTGCTTAAAGTTGGTTTTATTG 690 ASD_FRT_86_116_R TGAGATGTCGAAAAAAACGTT 1164 GTTGGCG GGCAAAATAC 1101 ASD_FRT_43_76_F TCAGTTTTAATGTCTCGTATGA 295 ASD_FRT_129_156_R TCCATATTGTTGCATAAAACC 1009 TCGAATCAAAAG TGTTGGC 1102 GALE_FRT_168_199_F TTATCAGCTAGACCTTTTAGGT 658 GALE_FRT_241_269_R TCACCTACAGCTTTAAAGCCA 973 AAAGCTAAGC GCAAAATG 1103 GALE_FRT_834_865_F TCAAAAAGCCCTAGGTAAAGAG 245 GALE_FRT_901_925_R TAGCCTTGGCAACATCAGCAA 915 ATTCCATATC AACT 1104 GALE_FRT_308_339_F TCCAAGGTACACTAAACTTACT 306 GALE_FRT_390_422_R TCTTCTGTAAAGGGTGGTTTA 1136 TGAGCTAATG TTATTCATCCCA 1105 IPAH_SGF_258_277_F TGAGGACCGTGTCGCGCTCA 458 IPAH_SGF_301_327_R TCCTTCTGATGCCTGATGGAC 1055 CAGGAG 1106 IPAH_SGF_113_134_F TCCTTGACCGCCTTTCCGATAC 350 IPAH_SGF_172_191_R TTTTCCAGCCATGCAGCGAC 1441 1107 IPAH_SGF_462_486_F TCAGACCATGCTCGCAGAGAAA 271 IPAH_SGF_522_540_R TGTCACTCCCGACACGCCA 1322 CTT 1111 RNASEP_BRM_461_488_ TAAACCCCATCGGGAGCAAGAC 147 RNASEP_BRM_542_561_R TGCCTCGCGCAACCTACCCG 1227 F CGAATA 1112 RNASEP_BRM_325_347_ TACCCCAGGGAAAGTGCCACAG 185 RNASEP_BRM_402_428_R TCTCTTACCCCACCCTTTCAC 1125 F A CCTTAC 1128 HUPB_CJ_113_134_F TAGTTGCTCAAACAGCTGGGCT 230 HUPB_CJ_157_188_R TCCCTAATAGTAGAAATAACT 1028 GCATCAGTAGC 1129 HUPB_CJ_76102_F TCCCGGAGCTTTTATGACTAAA 324 HUPB_CJ_157_188_R TCCCTAATAGTAGAAATAACT 1028 GCAGAT GCATCAGTAGC 1130 HUPB_CJ_76_102_F TCCCGGAGCTTTTATGACTAAA 324 HUPB_CJ_114_135_R TAGCCCAGCTGTTTGAGCAAC 913 GCAGAT T 1151 AB_MLST-11-OIF007_ TGAGATTGCTGAACATTTAATG 454 AB_MLST-11-OIF007_ TTGTACATTTGAAACAATATG 1418 62_91_F CTGATTGA 169_203_R CATGACATGTGAAT 1152 AB_MLST-11-OIF007_ TATTGTTTCAAATGTACAAGGT 243 AB_MLST-11-OIF007_ TCACAGGTTCTACTTCATCAA 969 185_214_F GAAGTGCG 291_324_R TAATTTCCATTGC 1153 AB_MLST-11-OIF007_ TGGAACGTTATCAGGTGCCCCA 541 AB_MLST-11-OIF007_ TTGCAATCGACATATCCATTT 1400 260_289_F AAAATTCG 364_393_R CACCATGCC 1154 AB_MLST-11-OIF007_ TGAAGTGCGTGATGATATCGAT 436 AB_MLST-11-OIF007_ TCCGCCAAAAACTCCCCTTTT 1036 206_239_F GCACTTGATGTA 318_344_R CACAGG 1155 AB_MLST-11-OIF007_ TCGGTTTAGTAAAAGAACGTAT 378 AB_MLST-11-OIF007_ TTCTGCTTGAGGAATAGTGCG 1392 522_552_F TGCTCAACC 587_610_R TGG 1156 AB_MLST-11-OIF007_ TCAACCTGACTGCGTGAATGGT 250 AB_MLST-11-OIF007_ TACGTTCTACGATTTCTTCAT 902 547_571_F TGT 656_686_R CAGGTACATC 1157 AB_MLST-11-OIF007_ TCAAGCAGAAGCTTTGGAAGAA 256 AB_MLST-11-OIF007_ TACAACGTGATAAACACGACC 881 601_627_F GAAGG 710_736_R AGAAGC 1158 AB_MLST-11-OIF007_ TCGTGCCCGCAATTTGCATAAA 384 AB_MLST-11-OIF007_ TAATGCCGGGTAGTGCAATCC 878 1202_1225_F GC 1266_1296_R ATTCTTCTAG 1159 AB_MLST-11-OIF007_ TCGTGCCCGCAATTTGCATAAA 384 AB_MLST-11-OIF007_ TGCACCTGCGGTCGAGCG 1199 1202_1225_F GC 1299_1316_R 1160 AB_MLST-11-OIF007_ TTGTAGCACAGCAAGGCAAATT 694 AB_MLST-11-OIF007_ TGCCATCCATAATCACGCCAT 1215 1234_1264_F TCCTGAAAC 1335_1362_R ACTGACG 1161 AB_MLST-11-OIF007_ TAGGTTTACGTCAGTATGGCGT 225 AB_MLST-11-OIF007_ TGCCAGTTTCCACATTTCACG 1212 1327_1356_F GATTATGG 1422_1448_R TTCGTG 1162 AB_MLST-11-OIF007_ TCGTGATTATGGATGGCAACGT 383 AB_MLST-11-OIF007_ TCGCTTGAGTGTAGTCATGAT 1083 1345_1369_F GAA 1470_1494_R TGCG 1163 AB_MLST-11-OIF007_ TTATGGATGGCAACGTGAAACG 662 AB_MLST_11-OIF007_ TCGCTTGAGTGTAGTCATGAT 1083 1351_1375_F CGT 1470_1494_R TGCG 1164 AB_MLST-11-OIF007_ TCTTTGCCATTGAAGATGACTT 422 AB_MLST-11-OIF007_ TCGCTTGAGTGTAGTCATGAT 1083 1387_1412_F AAGC 1470_1494_R TGCG 1165 AB_MLST-11-OIF007_ TACTAGCGGTAAGCTTAAACAA 194 AB_MLST-11-OIF007_ TGAGTCGGGTTCACTTTACCT 1173 1542_1569_F GATTGC 1656_1680_R GGCA 1166 AB_MLST-11-OIF007_ TTGCCAATGATATTCGTTGGTT 684 AB_MLST-11-OIF007_ TGAGTCGGGTTCACTTTACCT 1173 1566_1593_F AGCAAG 1656_1680_R GGCA 1167 AB_MLST-11-OIF007_ TCGGCGAAATCCGTATTCCTGA 375 AB_MLST-11-OIF007_ TACCGGAAGCACCAGCGACAT 890 1611_1638_F AAATGA 1731_1757_R TAATAG 1168 AB_MLST-11-OIF007_ TACCACTATTAATGTCGCTGGT 182 AB_MLST-11-OIF007_ TGCAACTGAATAGATTGCAGT 1195 1726_1752_F GCTTC 1790_1821_R AAGTTATAAGC 1169 AB_MLST-11-OIF007_ TTATAACTTACTGCAATCTATT 656 AB_MLST-11-OIF007_ TGAATTATGCAAGAAGTGATC 1151 1792_1826_F CAGTTGCTTGGTG 1876_1909_R AATTTTCTCACGA 1170 AB_MLST-11-OIF007_ TTATAACTTACTGCAATCTATT 656 AB_MLST-11-OIF007_ TGCCGTAACTAACATAAGAGA 1224 1792_1826_F CAGTTGCTTGGTG 1895_1927_R ATTATGCAAGAA 1171 AB_MLST-11-OIF007_ TGGTTATGTACCAAATACTTTG 618 AB_MLST-11-OIF007_ TGACGGCATCGATACCACCGT 1157 1970_2002_F TCTGAAGATGG 2097_2118_R C 1172 RNASEP_BRM_461_488_ TAAACCCCATCGGGAGCAAGAC 147 RNASEP_BRM_542_561_2_ TGCCTCGTGCAACCCACCCG 1228 F CGAATA R 2000 CTXB_NC002505_46_ TCAGCGTATGCACATGGAACTC 278 CTXB_NC002505_132_ TCCGGCTAGAGATTCTGTATA 1039 70_F CTC 162_R CGACAATATC 2001 FUR_NC002505_87_ TGAGTGCCAACATATCAGTGCT 465 FUR_NC002505_205_228_ TCCGCCTTCAAAATGGTGGCG 1037 113_F GAAGA R AGT 2002 FUR_NC002505_87_ TGAGTGCCAACATATCAGTGCT 465 FUR_NC002505_178_205_ TCACGATACCTGCATCATCAA 974 113_F GAAGA R ATTGGTT 2003 GAPA_NC002505_533_ TCGACAACACCATTATCTATGG 356 GAPA_NC002505_646_ TCAGAATCGATGCCAAATGCG 980 560_F TGTGAA 671_R TCATC 2004 GAPA_NC002505_694_ TCAATGAACGACCAACAAGTGA 259 GAPA_NC002505_769_ TCCTCTATGCAACTTAGTATC 1046 721_F TTGATG 798_R AACAGGAAT 2005 GAPA_NC002505_753_ TGCTAGTCAATCTATCATTCCG 517 GAPA_NC002505_856_ TCCATCGCAGTCACGTTTACT 1011 782_F GTTGATAC 881_R GTTGG 2006 GYRB_NC002505_2_ TGCCGGACAATTACGATTCATC 501 GYRB_NC002505_109_ TCCACCACCTCAAAGACCATG 1003 32_F GAGTATTAA 134_R TGGTG 2007 GYRB_NC002505_123_ TGAGGTGGTGGATAACTCAATT 460 GYRB_NC002505_199_ TCCGTCATCGCTGACAGAAAC 1042 152_F GATGAAGC 225_R TGAGTT 2008 GYRB_NC002505_768_ TATGCAGTGGAACGATGGTTTC 236 GYRB_NC002505_832_ TGGAAACCGGCTAAGTGAGTA 1262 794_F CAAGA 860_R CCACCATC 2009 GYRB_NC002505_837_ TGGTACTCACTTAGCGGGTTTC 603 GYRB_NC002505_937_ TCCTTCACGCGCATCATCACC 1054 860_F CG 957_R 2010 GYRB_NC002505_934_ TCGGGTGATGATGCGCGTGAAG 377 GYRB_NC002505_982_ TGGCTTGAGAATTTAGGATCC 1283 956_F G 1007_R GGCAC 2011 GYRB_NC002505_1161_ TAAAGCCCGTGAAATGACTCGT 148 GYRB_NC002505_1255_ TGAGTCACCCTCCACAATGTA 1172 1190_F CGTAAAGG 1284_R TAGTTCAGA 2012 OMPU_NC002505_85_ TACGCTGACGGAATCAACCAAA 190 OMPU_NC002505_154_ TGCTTCAGCACGGCCACCAAC 1254 110_F GCGG 180_R TTCTAG 2013 OMPU_NC002505_258_ TGACGGCCTATACGGTGTTGGT 451 OMPU_NC002505_346_ TCCGAGACCAGCGTAGGTGTA 1033 283_F TTCT 369_R ACG 2014 OMPU_NC002505_431_ TCACCGATATCATGGCTTACCA 266 OMPU_NC002505_544_ TCGGTCAGCAAAACGGTAGCT 1094 455_F CGG 567_R TGC 2015 OMPU_NC002505_533_ TAGGCGTGAAAGCAAGCTACCG 223 OMPU_NC002505_625_ TAGAGAGTAGCCATCTTCACC 908 557_F TTT 651_R GTTGTC 2016 OMPU_NC002505_689_ TAGGTGCTGGTTACGCAGATCA 224 OMPU_NC002505_725_ TGGGGTAAGACGCGGCTAGCA 1291 713_F AGA 751_R TGTATT 2017 OMPU_NC002505_727_ TACATGCTAGCCGCGTCTTAC 181 OMPU_NC002505_811_ TAGCAGCTAGCTCGTAACCAG 911 747_F 835_R TGTA 2018 OMPU_NC002505_931_ TACTACTTCAAGCCGAACTTCC 193 OMPU_NC002505_1033_ TTAGAAGTCGTAACGTGGACC 1368 953_F G 1053_R 2019 OMPU_NC002505_927_ TACTTACTACTTCAAGCCGAAC 197 OMPU_NC002505_1033_ TGGTTAGAAGTCGTAACGTGG 1307 953_F TTCCG 1054_R ACC 2020 TCPA_NC002505_48_ TCACGATAAGAAAACCGGTCAA 269 TCPA_NC002505_148_ TTCTGCGAATCAATCGCACGC 1391 73_F GAGG 170_R TG 2021 TDH_NC004605_265_ TGGCTGACATCCTACATGACTG 574 TDH_NC004605_357_386_ TGTTGAAGCTGTACTTGACCT 1351 289_F TGA R GATTTTACG 2022 VVHA_NC004460_772_ TCTTATTCCAACTTCAAACCGA 412 VVHA_NC004460_862_ TACCAAAGCGTGCACGATAGT 887 802_F ACTATGACG 886_R TGAG 2023 23S_EC_2643_2667_F TGCCTGTTCTTAGTACGAGAGG 508 23S_EC_2746_2770_R TGGGTTTCGCGCTTAGATGCT 1297 ACC TTCA 2024 16S_EC_713_732_ TAGAACACCGATGGCGAAGGC 202 16S_EC_789_811_R TGCGTGGACTACCAGGGTATC 1240 TMOD_F TA 2025 16S_EC_784_806_F TGGATTAGAGACCCTGGTAGTC 560 16S_EC_880_897_TMOD_R TGGCCGTACTCCCCAGGCG 1278 C 2026 16S_EC_959_981_F TGTCGATGCAACGCGAAGAACC 634 16S_EC_1052_1074_R TACGAGCTGACGACAGCCATG 896 T CA 2027 TUFB_EC_956_979_F TGCACACGCCGTTCTTCAACAA 489 TUFB_EC_1034_1058_2_R TGCATCACCATTTCCTTGTCC 1204 CT TTCG 2028 RPOC_EC_2146_2174_ TCAGGAGTCGTTCAACTCGATC 284 RPOC_EC_2227_2249_R TGCTAGGCCATCAGGCCACGC 1244 TMOD_F TACATGAT AT 2029 RPOB_EC_1841_1866_F TGGTTATCGCTCAGGCGAACTC 617 RPOB_EC_1909_1929_ TGCTGGATTCGCCTTTGCTAC 1250 CAAC TMOD_R G 2030 RPLB_EC_650_679_ TGACCTACAGTAAGAGGTTCTG 449 RPLB_EC_739_763_R TGCCAAGTGCTGGTTTACCCC 1208 TMOD_F TAATGAACC ATGG 2031 RPLB_EC_690_710_F TCCACACGGTGGTGGTGAAGG 309 RPLB_EC_737_760_R TGGGTGCTGGTTTACCCCATG 1295 GAG 2032 INFB_EC_1366_1393_F TCTCGTGGTGCACAAGTAACGG 397 INFB_EC_1439_1469_R TGTGCTGCTTTCGCATGGTTA 1335 ATATTA ATTGCTTCAA 2033 VALS_EC_1105_1124_ TCGTGGCGGCGTGGTTATCGA 385 VALS_EC_1195_1219_R TGGGTACGAACTGGATGTCGC 1292 TMOD_F CGTT 2034 SSPE_BA_113_137_F TGCAAGCAAACGCACAATCAGA 482 SSPE_BA_197_222_TMOD_ TTGCACGTCTGTTTCAGTTGC 1402 AGC R AAATTC 2035 RPOC_EC_2218_2241_ TCTGGCAGGTATGCGTGGTCTG 405 RPOC_EC_2313_2338_R TGGCACCGTGGGTTGAGATGA 1273 TMOD_F ATG AGTAC 2056 MECI-R_NC003923- TTTACACATATCGTGAGCAATG 698 MECI-_R_NC003923- TTGTGATATGGAGGTGTAGAA 1420 41798-_41609_33_60_ AACTGA 41798-41609_86_113_R GGTGTTA F 2057 AGR-III_NC003923- TCACCAGTTTGCCACGTATCTT 263 AGR-III_NC003923- ACCTGCATCCCTAAACGTACT 730 2108074-2109507_1_ CAA 2108074-2109507_56_ TGC 23_F 79_R 2058 AGR-III_NC003923- TGAGCTTTTAGTTGACTTTTTC 457 AGR-III_NC003923- TACTTCAGCTTCGTCCAATAA 906 2108074-2109507_ AACAGC 2108074-2109507_622_ AAAATCACAAT 569_596_F 653_R 2059 AGR-III_NC003923- TTTCACACAGCGTGTTTATAGT 701 AGR-III_NC003923- TGTAGGCAAGTGCATAAGAAA 1319 2108074-2109507_ TCTACCA 2108074-2109507_ TTGATACA 1024_1052_F 1070_1098_R 2060 AGR-I_AJ617706_622_ TGGTGACTTCATAATGGATGAA 610 AGR-I_AJ617706_694_ TCCCCATTTAATAATTCCACC 1021 651_F GTTGAAGT 726_R TACTATCACACT 2061 AGR-I_AJ617706_580_ TGGGATTTTAAAAAACATTGGT 579 AGR-I_AJ617706_626_ TGGTACTTCAACTTCATCCAT 1302 611_F AACATCGCAG 655_R TATGAAGTC 2062 AGR-II_NC002745- TCTTGCAGCAGTTTATTTGATG 415 AGR-II_NC002745- TTGTTTATTGTTTCCATATGC 1424 2079448-2080879_ AACCTAAAGT 2079448-2080879_ TACACACTTTC 620_651_F 700_731_R 2063 AGR-II_NC002745- TGTACCCGCTGAATTAACGAAT 624 AGR-II_NC002745- TCGCCATAGCTAAGTTGTTTA 1077 2079448-2080879_ TTATACGAC 2079448-2080879_ TTGTTTCCAT 649_679_F 715_745_R 2064 AGR-IV_AJ617711_ TGGTATTCTATTTTGCTGATAA 606 AGR-IV_AJ617711_ TGCGCTATCAACGATTTTGAC 1233 931_961_F TGACCTCGC 1004_1035_R AATATATGTGA 2065 AGR-IV_AJ617711_ TGGCACTCTTGCCTTTAATATT 562 AGR-IV_AJ617711_ TCCCATACCTATGGCGATAAC 1017 250_283_F AGTAAACTATCA 309_335_R TGTCAT 2066 BLAZ_NC002952 TCCACTTATCGCAAATGGAAAA 312 BLAZ_NC002952 TGGCCACTTTTATCAGCAACC 1277 (1913827 . . . TTAAGCAA (1913827 . . . TTACAGTC 1914672)_68_68_F 1914672)_68_68_R 2067 BLAZ_NC002952 TGCACTTATCGCAAATGGAAAA 494 BLAZ_NC002952 TAGTCTTTTGGAACACCGTCT 926 (1913827 . . . TTAAGCAA (1913827 . . . TTAATTAAAGT 1914672)_68_68_2_F 1914672)_68_68_2_R 2068 BLAZ_NC002952 TGATACTTCAACGCCTGCTGCT 467 BLAZ_NC002952 TGGAACACCGTCTTTAATTAA 1263 (1913827 . . . TTC (1913827 . . . AGTATCTCC 1914672)_68_68_3_F 1914672)_68_68_3_R 2069 BLAZ_NC002952 TATACTTCAACGCCTGCTGCTT 232 BLAZ_NC002952 TCTTTTCTTTGCTTAATTTTC 1145 (1913827 . . . TC (1913827 . . . CATTTGCGAT 1914672)_68_68_4_F 1914672)_68_68_4_R 2070 BLAZ_NC002952 TGCAATTGCTTTAGTTTTAAGT 487 BLAZ_NC002952 TTACTTCCTTACCACTTTTAG 1366 (1913827 . . . GCATGTAATTC (1913827 . . . TATCTAAAGCATA 1914672)_1_33_F 1914672)_34_67_R 2071 BLAZ_NC002952 TCCTTGCTTTAGTTTTAAGTGC 351 BLAZ_NC002952 TGGGGACTTCCTTACCACTTT 1289 (1913827 . . . ATGTAATTCAA (1913827 . . . TAGTATCTAA 1914672)_3_34_F 1914672)_40_68_R 2072 BSA-A_NC003923- TAGCGAATGTGGCTTTACTTCA 214 BSA-A_NC003923- TGCAAGGGAAACCTAGAATTA 1197 1304065- CAATT 1304065- CAAACCCT 1303589_99_125_F 1303589_165_193_R 2073 BSA-A_NC003923- ATCAATTTGGTGGCCAAGAACC 32 BSA-A_NC003923- TGCATAGGGAAGGTAACACCA 1203 1304065- TGG 1304065- TAGTT 1303589_194_218_F 1303589_253_278_R 2074 BSA-A_NC003923- TTGACTGCGGCACAACACGGAT 679 BSA-A_NC003923- TAACAACGTTACCTTCGCGAT 856 1304065- 1304065- CCACTAA 1303589_328_349_F 1303589_388_415_R 2075 BSA-A_NC003923- TGCTATGGTGTTACCTTCCCTA 519 BSA-A_NC003923- TGTTGTGCCGCAGTCAAATAT 1353 1304065- TGCA 1304065- CTAAATA 1303589_253_278_F 1303589_317_344_R 2076 BSA-B_NC003923- TAGCAACAAATATATCTGAAGC 209 BSA-B_NC003923- TGTGAAGAACTTTCAAATCTG 1331 1917149- AGCGTACT 1917149- TGAATCCA 1914156_953_982_F 1914156_1011_1039_R 2077 BSA-B_NC003923- TGAAAAGTATGGATTTGAACAA 426 BSA-B_NC003923- TCTTCTTGAAAAATTGTTGTC 1138 1917149- CTCGTGAATA 1917149- CCGAAAC 1914156_1050_1081_F 1914156_1109_1136_R 2078 BSA-B_NC003923- TCATTATCATGCGCCAATGAGT 300 BSA-B_NC003923- TGGACTAATAACAATGAGCTC 1267 1917149- GCAGA 1917149- ATTGTACTGA 1914156_1260_1286_F 1914156_1323_1353_R 2079 BSA-B_NC003923- TTTCATCTTATCGAGGACCCGA 703 BSA-B_NC003923- TGAATATGTAATGCAAACCAG 1148 1917149- AATCGA 1917149- TCTTTGTCAT 1914156_2126_2153_F 1914156_2186_2216_R 2080 ERMA_NC002952- TCGCTATCTTATCGTTGAGAAG 372 ERMA_NC002952-55890- TGAGTCTACACTTGGCTTAGG 1174 55890- GGATT 56621_487_513_R ATGAAA 56621_366_392_F 2081 ERMA_NC002952- TAGCTATCTTATCGTTGAGAAG 217 ERMA_NC002952-55890- TGAGCATTTTTATATCCATCT 1167 55890- GGATTTGC 56621_438_465_R CCACCAT 56621_366_395_F 2082 ERMA_NC002952- TGATCGTTGAGAAGGGATTTGC 470 ERMA_NC002952-55890- TCTTGGCTTAGGATGAAAATA 1143 55890- GAAAAGA 56621_473_504_R TAGTGGTGGTA 56621_374_402_F 2083 ERMA_NC002952- TGCAAAATCTGCAACGAGCTTT 480 ERMA_NC002952-55890- TCAATACAGAGTCTACACTTG 964 55890- GG 56621_491_520_R GCTTAGGAT 56621_404_427_F 2084 ERMA_NC002952- TCATCCTAAGCCAAGTGTAGAC 297 ERMA_NC002952-55890- TGGACGATATTCACGGTTTAC 1266 55890- TCTGTA 56621_586_615_R 56621_489_516_F 2085 ERMA_NC002952- TATAAGTGGGTAAACCGTGAAT 231 ERMA_NC002952-55890- TTGACATTTGCATGCTTCAAA 1397 55890- ATCGTGT 56621_640_665_R GCCTG 56621_586_614_F 2086 ERMC_NC005908-2004- TCTGAACATGATAATATCTTTG 399 ERMC_NC005908-2004- TCCGTAGTTTTGCATAATTTA 1041 2738_85_116_F AAATCGGCTC 2738_173-206-4 TGGTCTATTTCAA 2087 ERMC_NC005908-2004- TCATGATAATATCTTTGAAATC 298 ERMC_NC005908-2004- TTTATGGTCTATTTCAATGGC 1429 2738_90_120_F GGCTCAGGA 2738_160_189_R AGTTACGAA 2088 ERMC_NC005908-2004- TCAGGAAAAGGGCATTTTACCC 283 ERMC_NC005908-2004- TATGGTCTATTTCAATGGCAG 936 2738_115_139_F TTG 2738_161_187_R TTACGA 2089 ERMC_NC005908-2004- TAATCGTGGAATACGGGTTTGC 168 ERMC_NC005908-2004- TCAACTTCTGCCATTAAAAGT 956 2738_374_397_F TA 2738_425_452_R AATGCCA 2090 ERMC_NC005908-2004- TCTTTGAAATCGGCTCAGGAAA 421 ERMC_NC005908-2004- TGATGGTCTATTTCAATGGCA 1185 2738_101_125_F AGG 2738_159_188_R GTTACGAAA 2091 ERMB_Y13600-625- TGTTGGGAGTATTCCTTACCAT 644 ERMB_Y13600-625- TCAACAATCAGATAGATGTCA 953 1362_291_321_F TTAAGCACA 1362_352_380_R GACGCATG 2092 ERMB_Y13600-625- TGGAAAGCCATGCGTCTGACAT 536 ERMB_Y13600-625- TGCAAGAGCAACCCTAGTGTT 1196 1362_344_367_F CT 1362_415_437_R CG 2093 ERMB_Y13600-625- TGGATATTCACCGAACACTAGG 556 ERMB_Y13600-625- TAGGATGAAAGCATTCCGCTG 919 1362_404_429_F GTTG 1362_471_493_R GC 2094 ERMB_Y13600-625- TAAGCTGCCAGCGGAATGCTTT 161 ERMB_Y13600-625- TCATCTGTGGTATGGCGGGTA 989 1362_465_487_F C 1362_521_545_R AGTT 2095 PVLUK_NC003923- TGAGCTGCATCAACTGTATTGG 456 PVLUK_NC003923- TGGAAAACTCATGAAATTAAA 1261 1529595- ATAG 1529595- GTGAAAGGA 1531285_688_713_F 1531285_775_804_R 2096 PVLUK_NC003923- TGGAACAAAATAGTCTCTCGGA 539 PVLUK_NC003923- TCATTAGGTAAAATGTCTGGA 993 1529595- TTTTGACT 1529595- CATGATCCAA 1531285_1039_106_F 1531285_1095_1125_R 2097 PVLUK_NC003923- TGAGTAACATCCATATTTCTGC 461 PVLUK_NC003923- TCTCATGAAAAAGGCTCAGGA 1124 1529595- CATACGT 1529595- GATACAAG 1531285_908_936_F 1531285_950_978_R 2098 PVLUK_NC003923- TCGGAATCTGATGTTGCAGTTG 373 PVLUK_NC003923- TCACACCTGTAAGTGAGAAAA 968 1529595- TT 1529595- AGGTTGAT 1531285_610_633_F 1531285_654_682_R 2099 SA442_NC003923- TGTCGGTACACGATATTCTTCA 635 SA442_NC003923- TTTCCGATGCAACGTAATGAG 1433 2538576- CGA 2538576- ATTTCA 2538831_11_35_F 2538831_98_124_R 2100 SA442_NC003923- TGAAATCTCATTACGTTGCATC 427 SA442_NC003923- TCGTATGACCAGCTTCGGTAC 1098 2538576- GGAAA 2538576- TACTA 2538831_98_124_F 2538831_163_188_R 2101 SA442_NC003923- TCTCATTACGTTGCATCGGAAA 395 SA442_NC003923- TTTATGACCAGCTTCGGTACT 1428 2538576- CA 2538576- ACTAAA 2538831_103_126_F 2538831_161_187_R 2102 SA442_NC003923- TAGTACCGAAGCTGGTCATACG 226 SA442_NC003923- TGATAATGAAGGGAAACCTTT 1179 2538576- A 2538576- TTCACG 2538831_166_188_F 2538831_231_257_R 2103 SEA_NC003923- TGCAGGGAACAGCTTTAGGCA 495 SEA_NC003923- TCGATCGTGACTCTCTTTATT 1070 2052219- 2052219- TTCAGTT 2051456_115_135_F 2051456_173_200_R 2104 SEA_NC003923- TAACTCTGATGTTTTTGATGGG 156 SEA_NC003923- TGTAATTAACCGAAGGTTCTG 1315 2052219- AAGGT 2052219- TAGAAGTATG 2051456_572_598_F 2051456_621_651_R 2105 SEA_NC003923- TGTATGGTGGTGTAACGTTACA 629 SEA_NC003923- TAACCGTTTCCAAAGGTACTG 861 2052219- TGATAATAATC 2052219- TATTTTGT 2051456_382_414_F 2051456_464_492_R 2106 SEA_NC003923- TTGTATGTATGGTGGTGTAACG 695 SEA_NC003923- TAACCGTTTCCAAAGGTACTG 862 2052219- TTACATGA 2052219- TATTTTGTTTACC 2051456_377_406_F 2051456_459_492_R 2107 SEB_NC002758- TTTCACATGTAATTTTGATATT 702 SEB_NC002758- TCATCTGGTTTAGGATCTGGT 988 2135540- CGCACTGA 2135540- TGACT 2135140_208_237_F 2135140_273_298_R 2108 SEB_NC002758- TATTTCACATGTAATTTTGATA 244 SEB_NC002758- TGCAACTCATCTGGTTTAGGA 1194 2135540- TTCGCACT 2135540- TCT 2135140_206_235_F 2135140_281_304_R 2109 SEB_NC002758- TAACAACTCGCCTTATGAAACG 151 SEB_NC002758- TGTGCAGGCATCATGTCATAC 1334 2135540- GGATATA 2135540- CAA 2135140_402_402_F 2135140-402-402-R 2110 SEB_NC002758- TTGTATGTATGGTGGTGTAACT 696 SEB_NC002758- TTACCATCTTCAAATACCCGA 1361 2135540- GAGCA 2135540- ACAGTAA 2135140_402_402_F 2135140_402_402_2_R 2111 SEC_NC003923- TTAACATGAAGGAAACCACTTT 648 SEC_NC003923-851678- TGAGTTTGCACTTCAAAAGAA 1177 851678- GATAATGG 852768_620_647_R ATTGTGT 852768_546_575_F 2112 SEC_NC003923- TGGAATAACAAAACATGAAGGA 546 SEC_NC003923_851678- TCAGTTTGCACTTCAAAAGAA 985 851678- AACCACTT 852768_619_647_R ATTGTGTT 852768_537_566_F 2113 SEC_NC003923- TGAGTTTAACAGTTCACCATAT 466 SEC_NC003923-851678- TCGCCTGGTGCAGGCATCATA 1078 851678- GAAACAGG 852768_794_815_R T 852768_720_749_F 2114 SEC_NC003923- TGGTATGATATGATGCCTGCAC 604 SEC_NC003923-851678- TCTTCACACTTTTAGAATCAA 1133 851678- CA 852768_853_886_R CCGTTTTATTGTC 852768_787_810_F 2115 SED_M28521_657_682_ TGGTGGTGAAATAGATAGGACT 615 SED_M28521_741_770_R TGTACACCATTTATCCACAAA 1318 F GCTT TTGATTGGT 2116 SED_M28521_690_711_ TGGAGGTGTCACTCCACACGAA 554 SED_M28521_739_770_R TGGGCACCATTTATCCACAAA 1288 F TTGATTGGTAT 2117 SED_M28521_833_854_ TTGCACAAGCAAGGCGCTATTT 683 SED_M28521_888_991_R TCGCGCTGTATTTTTCCTCCG 1079 F AGA 2118 SED_M28521_962_987_ TGGATGTTAAGGGTGATTTTCC 559 SED_M28521_1022_1048_ TGTCAATATGAAGGTGCTCTG 1320 F CGAA R TGGATA 2119 SEA-SEE_NC002952- TTTACACTACTTTTATTCATTG 699 SEA-SEE_NC002952- TCATTTATTTCTTCGCTTTTC 994 2131289- CCCTAACG 2131289- TCGCTAC 2130703_16_45_F 2130703_71_98_R 2120 SEA-SEE_NC002952 TGATCATCCGTGGTATAACGAT 469 SEA-SEE_NC002952- TAAGCACCATATAAGTCTACT 870 2131289- TTATTAGT 2131289- TTTTTCCCTT 2130703_249_278_F 2130703_314_344_R 2121 SEE_NC002952- TGACATGATAATAACCGATTGA 445 SEE_NC002952- TCTATAGGTACTGTAGTTTGT 1120 2131289- CCGAAGA 2131289- TTTCCGTCT 2130703_409_437_F 2130703_465_494_R 2122 SEE_NC002952- TGTTCAAGAGCTAGATCTTCAG 640 SEE_NC002952- TTTGCACCTTACCGCCAAAGC 1436 2131289- GCAA 2131289- T 2130703_525_550_F 2130703_586_586_R 2123 SEE_NC002952- TGTTCAAGAGCTAGATCTTCAG 639 SEE_NC002952- TACCTTACCGCCAAAGCTGTC 892 2131289- GCA 2131289- T 2130703_525_549_F 2130703_586_586_2_R 2124 SEE_NC002952- TCTGGAGGCACACCAAATAAAA 403 SEE_NC002952- TCCGTCTATCCACAAGTTAAT 1043 2131289- CA 2131289- TGGTACT 2130703_361_384_F 2130703_444_471_R 2125 SEG_NC002758- TGCTCAACCCGATCCTAAATTA 520 SEG_NC002758- TAACTCCTCTTCCTTCAACAG 863 1955100- GACGA 1955100- GTGGA 1954171_225_251_F 1954171_321_346_R 2126 SEG_NC002758- TGGACAATAGACAATCACTTGG 548 SEG_NC002758- TGCTTTGTAATCTAGTTCCTG 1260 1955100- ATTTACA 1955100- AATAGTAACCA 1954171_623_651_F 1954171_671_702_R 2127 SEG_NC002758- TGGAGGTTGTTGTATGTATGGT 555 SEG_NC002758- TGTCTATTGTCGATTGTTACC 1329 1955100- GGT 1955100- TGTACAGT 1954171_540_564_F 1954171_607_635_R 2128 SEG_NC002758- TACAAAGCAAGACACTGGCTCA 173 SEG_NC002758- TGATTCAAATGCAGAACCATC 1187 1955100- CTA 1955100- AAACTCG 1954171_694_718_F 1954171_735_762_R 2129 SEH_NC002953- TTGCAACTGCTGATTTAGCTCA 682 SEH_NC002953-60024- TAGTGTTGTACCTCCATATAG 927 60024- GA 60977_547_576_R ACATTCAGA 60977_449_472_F 2130 SEH_NC002953- TAGAAATCAAGGTGATAGTGGC 201 SEH_NC002953-60024- TTCTGAGCTAAATCAGCAGTT 1390 60024- AATGA 60977_450_473_R GCA 60977_408_434_F 2131 SEH_NC002953- TCTGAATGTCTATATGGAGGTA 400 SEH_NC002953-60024- TACCATCTACCCAAACATTAG 888 60024- CAACACTA 60977_608_634_R CACCAA 60977_547_576_F 2132 SEH_NC002953- TTCTGAATGTCTATATGGAGGT 677 SEH_NC002953-60024- TAGCACCAATCACCCTTTCCT 909 60024- ACAACACT 60977_594_616_R GT 60977_546_575_F 2133 SEI_NC002758- TCAACTCGAATTTTCAACAGGT 253 SEI_NC002758- TCACAAGGACCATTATAATCA 966 1957830- ACCA 1957830- ATGCCAA 1956949_324_349_F 1956949_419_446_R 2134 SEI_NC002758- TTCAACAGGTACCAATGATTTG 666 SEI_NC002758- TGTACAAGGACCATTATAATC 1316 1957830- ATCTCA 1957830- AATGCCA 1956949_336_363_F 1956949_420_447_R 2135 SEI_NC002758- TGATCTCAGAATCTAATAATTG 471 SEI_NC002758- TCTGGCCCCTCCATACATGTA 1129 1957830- GGACGAA 1957830- TTTAG 1956949_356_384_F 1956949_449_474_R 2136 SEI_NC002758- TCTCAAGGTGATATTGGTGTAG 394 SEI_NC002758- TGGGTAGGTTTTTATCTGTGA 1293 1957830- GTAACTTAA 1957830- CGCCTT 1956949_223_253_F 1956949_290_316_R 2137 SEJ_AF053140_1307_ TGTGGAGTAACACTGCATGAAA 637 SEJ_AF053140_1381_ TCTAGCGGAACAACAGTTCTG 1118 1332_F ACAA 1404_R ATG 2138 SEJ_AF053140_1378_ TAGCATCAGAACTGTTGTTCCG 211 SEJ_AF053140_1429_ TCCTGAAGATCTAGTTCTTGA 1049 1403_F CTAG 1458_R ATGGTTACT 2139 SEJ_AF053140_1431_ TAACCATTCAAGAACTAGATCT 153 SEJ_AF053140_1500_ TAGTCCTTTCTGAATTTTACC 925 1459_F TCAGGCA 1531_R ATCAAAGGTAC 2140 SEJ_AF053140_1434_ TCATTCAAGAACTAGATCTTCA 301 SEJ_AF053140_1521_ TCAGGTATGAAACACGATTAG 984 1461_F GGCAAG 1549_R TCCTTTCT 2141 TSST_NC002758- TGGTTTAGATAATTCCTTAGGA 619 TSST_NC002758- TGTAAAAGCAGGGCTATAATA 1312 2137564- TCTATGCGT 2137564- AGGACTC 2138293_206_236_F 2138293_278_305_R 2142 TSST_NC002758- TGCGTATAAAAAACACAGATGG 514 TSST_NC002758- TGCCCTTTTGTAAAAGCAGGG 1221 2137564- CAGCA 2137564- CTAT 2138293_232_258_F 2138293_289_313_R 2143 TSST_NC002758- TCCAAATAAGTGGCGTTACAAA 304 TSST_NC002758- TACTTTAAGGGGCTATCTTTA 907 2137564- TACTGAA 2137564- CCATGAACCT 2138293_382_410_F 2138293_448_478_R 2144 TSST_NC002758- TCTTTTACAAAAGGGGAAAAAG 423 TSST_NC002758- TAAGTTCCTTCGCTAGTATGT 874 2137564- TTGACTT 2137564- TGGCTT 2138293_297_325_F 2138293_347_373_R 2145 ARCC_NC003923- TCGCCGGCAATGCCATTGGATA 368 ARCC_NC003923- TGAGTTAAAATGCGATTGATT 1175 2725050- 2725050- TCAGTTTCCAA 2724595_37_58_F 2724595_97_128_R 2146 ARCC_NC003923- TGAATAGTGATAGAACTGTAGG 437 ARCC_NC003923- TCTTCTTCTTTCGTATAAAAA 1137 2725050- CACAATCGT 2725050- GGACCAATTGG 2724595_131_161_F 2724595_214_245_R 2147 ARCC_NC003923- TTGGTCCTTTTTATACGAAAGA 691 ARCC_NC003923- TGGTGTTCTAGTATAGATTGA 1306 2725050- AGAAGTTGAA 2725050- GGTAGTGGTGA 2724595_218_249_F 2724595_322_353_R 2148 AROE_NC003923- TTGCGAATAGAACGATGGCTCG 686 AROE_NC003923- TCGAATTCAGCTAAATACTTT 1064 1674726- T 1674726- TCAGCATCT 1674277_371_393_F 1674277_435_464_R 2149 AROE_NC003923- TGGGGCTTTAAATATTCCAATT 590 AROE_NC003923- TACCTGCATTAATCGCTTGTT 891 1674726- GAAGATTTTCA 1674726- CATCAA 1674277_30_62_F 1674277_155_181_R 2150 AROE_NC003923- TGATGGCAAGTGGATAGGGTAT 474 AROE_NC003923- TAAGCAATACCTTTACTTGCA 869 1674726- AATACAG 1674726- CCACCTG 1674277_204_232_F 1674277_308_335_R 2151 GLPF_NC003923- TGCACCGGCTATTAAGAATTAC 491 GLPF_NC003923- TGCAACAATTAATGCTCCGAC 1193 1296927- TTTGCCAACT 1296927- AATTAAAGGATT 1297391_270_301_F 1297391_382_414_R 2152 GLPF_NC003923- TGGATGGGGATTAGCGGTTACA 558 GLPF_NC003923- TAAAGACACCGCTGGGTTTAA 850 1296927- ATG 1296927- ATGTGCA 1297391_27_51_F 1297391_81_108_R 2153 GLPF_NC003923- TAGCTGGCGCGAAATTAGGTGT 218 GLPF_NC003923- TCACCGATAAATAAAATACCT 972 1296927- 1296927- AAAGTTAATGCCATTG 1297391_239_260_F 1297391_323_359_R 2154 GMK_NC003923- TACTTTTTTAAAACTAGGGATG 200 GMK_NC003923- TGATATTGAACTGGTGTACCA 1180 1190906- CGTTTGAAGC 1190906- TAATAGTTGCC 1191334_91_122_F 1191334_166_197_R 2155 GMK_NC003923- TGAAGTAGAAGGTGCAAAGCAA 435 GMK_NC003923- TCGCTCTCTCAAGTGATCTAA 1082 1190906- GTTAGA 1190906- ACTTGGAG 1191334_240_267_F 1191334_305_333_R 2156 GMK_NC003923- TCACCTCCAAGTTTAGATCACT 268 GMK_NC003923- TGGGACGTAATCGTATAAATT 1284 1190906- TGAGAGA 1190906- CATCATTTC 1191334_301_329_F 1191334_403_432_R 2157 PTA_NC003923- TCTTGTTTATGCTGGTAAAGCA 418 PTA_NC003923-628885- TGGTACACCTGGTTTCGTTTT 1301 628885- GATGG 629355_314_345_R GATGATTTGTA 629355_237_263_F 2158 PTA_NC003923- TGAATTAGTTCAATCATTTGTT 439 PTA_NC003923-628885- TGCATTGTACCGAAGTAGTTC 1207 628885- GAACGACGT 629355_211_239_R ACATTGTT 629355_141_171_F 2159 PTA_NC003923- TCCAAACCAGGTGTATCAAGAA 303 PTA_NC003923_628885- TGTTCTGGATTGATTGCACAA 1349 628885- CATCAGG 629355_393_422_R TCACCAAAG 629355_328_356_F 2160 TPI_NC003923- TGCAAGTTAAGAAAGCTGTTGC 486 TPI_NC003923-830671- TGAGATGTTGATGATTTACCA 1165 830671- AGGTTTAT 831072_209_239_R GTTCCGATTG 831072_131_160_F 2161 TPI_NC003923- TCCCACGAAACAGATGAAGAAA 318 TPI_NC003923-830671- TGGTACAACATCGTTAGCTTT 1300 830671- TTAACAAAAAAG 831072_97_129_R ACCACTTTCACG 831072_1_34_F 2162 TPI_NC003923- TCAAACTGGGCAATCGGAACTG 246 TPI_NC003923-830671- TGGCAGCAATAGTTTGACGTA 1275 830671- GTAAATC 831072_253_286_R CAAATGCACACAT 831072_199_227_F 2163 YQI_NC003923- TGAATTGCTGCTATGAAAGGTG 440 YQI_NC003923-378916- TCGCCAGCTAGCACGATGTCA 1076 378916- GCTT 379431_259_284_4 TTTTC 379431_142_167_F 2164 YQI_NC003923- TACAACATATTATTAAAGAGAC 175 YQI_NC003923-378916- TTCGTGCTGGATTTTGTCCTT 1388 378916- GGGTTTGAATCC 379431_120_145_R GTCCT 379431_44_77_F 2165 YQI_NC003923- TCCAGCACGAATTGCTGCTATG 314 YQI_NC003923-378916- TCCAACCCAGAACCACATACT 997 378916- AAAG 379431_193_221_R TTATTCAC 379431_135_160_F 2166 YQI_NC003923- TAGCTGGCGGTATGGAGAATAT 219 YQI_NC003923-378916- TCCATCTGTTAAACCATCATA 1013 378916- GTCT 379431_364_396_R TACCATGCTATC 379431_275_300_F 2167 BLAZ_(1913827 . . . TCCACTTATCGCAAATGGAAAA 312 BLAZ_(1913827 . . . TGGCCACTTTTATCAGCAACC 1277 1914672)_546_575_F TTAAGCAA 1914672)_655_683_R TTACAGTC 2168 BLAZ_(1913827 . . . TGCACTTATCGCAAATGGAAAA 494 BLAZ_(1913827 . . . TAGTCTTTTGGAACACCGTCT 926 1914672)_546_575_F TTAAGCAA 1914672)_628_659_R TTAATTAAAGT 2169 BLAZ_(1913827 . . . TGATACTTCAACGCCTGCTGCT 467 BLAZ_(1913827 . . . TGGAACACCGTCTTTAATTAA 1263 1914672)_507_531_F TC 1914672)_622_651_R AGTATCTCC 2170 BLAZ_(1913827 . . . TATACTTCAACGCCTGCTGCTT 232 BLAZ_(1913827 . . . TCTTTTCTTTGCTTAATTTTC 1145 1914672)_508_531_F TC 1914672)_553_583_R CATTTGCGAT 2171 BLAZ_(1913827 . . . TGCAATTGCTTTAGTTTTAAGT 487 BLAZ_(1913827 . . . TTACTTCCTTACCACTTTTAG 1366 1914672)_24_56_F GCATGTAATTC 1914672)_121_154_R TATCTAAAGCATA 2172 BLAZ_(1913827 . . . TCCTTGCTTTAGTTTTAAGTGC 351 BLAZ_(1913827 . . . TGGGGACTTCCTTACCACTTT 1289 1914672)_26_58_F ATGTAATTCAA 1914672)_127_157_R TAGTATCTAA 2173 BLAZ_NC002952- TCCACTTATCGCAAATGGAAAA 312 BLAZ_NC002952- TGGCCACTTTTATCAGCAACC 1277 1913827- TTAAGCAA 1913827- TTACAGTC 1914672_546_575_F 1914672_655_683_R 2174 BLAZ_NC002952- TGCACTTATCGCAAATGGAAAA 494 BLAZ_NC002952- TAGTCTTTTGGAACACCGTCT 926 1913827- TTAAGCAA 1913827- TTAATTAAAGT 1914672_546_575_F 1914672_628_659_R 2175 BLAZ_NC002952- TGATACTTCAACGCCTGCTGCT 467 BLAZ_NC002952- TGGAACACCGTCTTTAATTAA 1263 1913827- TTC 1913827- AGTATCTCC 1914672_507_531_F 1914672_622_651_R 2176 BLAZ_NC002952- TATACTTCAACGCCTGCTGCTT 232 BLAZ_NC002952- TCTTTTCTTTGCTTAATTTTC 1145 1913827- TC 1913827- CATTTGCGAT 1914672_508_531_F 1914672_553_583_R 2177 BLAZ_NC002952- TGCAATTGCTTTAGTTTTAAGT 487 BLAZ_NC002952- TTACTTCCTTACCACTTTTAG 1366 1913827- GCATGTAATTC 1913827- TATCTAAAGCATA 1914672_24_56_F 1914672_121_154_R 2178 BLAZ_NC002952- TCCTTGCTTTAGTTTTAAGTGC 351 BLAZ_NC002952- TGGGACTTCCTTACCACTTT 1289 1913827- ATGTAATTCAA 1913827- TAGTATCTAA 1914672_26_58_F 1914672_127_157_R 2247 TUFB_NC002758- TGTTGAACGTGGTCAAATCAAA 643 TUFB_NC002758- TGTCACCAGCTTCAGCGTAGT 1321 615038- GTTGGTG 615038- CTAATAA 616222_693_721_F 616222_793_820_R 2248 TUFB_NC002758- TCGTGTTGAACGTGGTCAAATC 386 TUFB_NC002758- TGTCACCAGCTTCAGCGTAGT 1321 615038- AAAGT 615038- CTAATAA 616222_690_716_F 616222_793_820_R 2249 TUFB_NC002758- TGAACGTGGTCAAATCAAAGTT 430 TUFB_NC002758- TGTCACCAGCTTCAGCGTAGT 1321 615038- GGTGAAGA 615038- CTAATAA 616222_696_725_F 616222_793_820_R 2250 TUFB_NC002758- TCCCAGGTGACGATGTACCTGT 320 TUFB_NC002758- TGGTTTGTCAGAATCACGTTC 1311 615038- AATC 615038- TGGAGTTGG 616222_488_513_F 616222_601_630_R 2251 TUFB_NC002758- TGAAGGTGGACGTCACACTCCA 433 TUFB_NC002758- TAGGCATAACCATTTCAGTAC 922 615038- TTCTTC 615038- CTTCTGGTAA 616222_945_972_F 616222_1030_1060_R 2252 TUFB_NC002758- TCCAATGCCACAAACTCGTGAA 307 TUFB_NC002758- TTCCATTTCAACTAATTCTAA 1382 615038- CA 615038- TAATTCTTCATCGTC 616222_333_356_F 616222_424_459_R 2253 NUC_NC002758- TCCTGAAGCAAGTGCATTTACG 342 NUC_NC002758-894288- TACGCTAAGCCACGTCCATAT 899 894288- A 894974_483_509_R TTATCA 894974_402_424_F 2254 NUC_NC002758- TCCTTATAGGGATGGCTATCAG 349 NUC_NC002758-894288- TGTTTGTGATGCATTTGCTGA 1354 894288- TAATGTT 894974_165_189_R GCTA 894974_53_81_F 2255 NUC_NC002758- TCAGCAAATGCATCACAAACAG 273 NUC_NC002758-894288- TAGTTGAAGTTGCACTATATA 928 894288- ATAA 894974_222_250_R CTGTTGGA 894974_169_194_F 2256 NUC_NC002758- TACAAAGGTCAACCAATGACAT 174 NUC_NC002758-894288- TAAATGCACTTGCTTCAGGGC 853 894288- TCAGACTA 894974_396_421_R CATAT 894974_316_345_F 2270 RPOB_EC_3798_3821_ TGGCCAGCGCTTCGGTGAAATG 566 RPOB_EC_3868_3895_R TCACGTCGTCCGACTTCACGG 979 1_F GA TCAGCAT 2271 RPOB_EC_3789_3812_F TCAGTTCGGCGGTCAGCGCTTC 294 RPOB_EC_3860_3890_R TCGTCGGACTTAACGGTCAGC 1107 GG ATTTCCTGCA 2272 RPOB_EC_3789_3812_F TCAGTTCGGCGGTCAGCGCTTC 294 RPOB_EC_3860_3890_2_R TCGTCCGACTTAACGGTCAGC 1102 GG ATTTCCTGCA 2273 RPOB_EC_3789_3812_F TCAGTTCGGCGGTCAGCGCTTC 294 RPOB_EC_3862_3890_R TCGTCGGACTTAACGGTCAGC 1106 GG ATTTCCTG 2274 RPOB_EC_3789_3812_F TCAGTTCGGCGGTCAGCGCTTC 294 RPOB_EC_3862_3890_2_R TCGTCCGACTTAACGGTCAGC 1101 GG ATTTCCTG 2275 RPOB_EC_3793_3812_F TTCGGCGGTCAGCGCTTCGG 674 RPOB_EC_3865_3890_R TCGTCGGACTTAACGGTCAGC 1105 ATTTC 2276 RPOB_EC_3793_3812_F TTCGGCGGTCAGCGCTTCGG 674 RPOB_EC_3865_3890_2_R TCGTCCGACTTAACGGTCAGC 1100 ATTTC 2309 MUPR_X75439_1658_ TCCTTTGATATATTATGCGATG 352 MUPR_X75439_1744_ TCCCTTCCTTAATATGAGAAG 1030 1689_F GAAGGTTGGT 1773_R GAAACCACT 2310 MUPR_X75439_1330_ TTCCTCCTTTTGAAAGCGACGG 669 MUPR_X75439_1413_ TGAGCTGGTGCTATATGAACA 1171 1353_F TT 1441_R ATACCAGT 2312 MUPR_X75439_1314_ TTTCCTCCTTTTGAAAGCGACG 704 MUPR_X75439_1381_ TATATGAACAATACCAGTTCC 931 1338_F GTT 1409_R TTCTGAGT 2313 MUPR_X75439_2486_ TAATTGGGCTCTTTCTCGCTTA 172 MUPR_X75439_2548_ TTAATCTGGCTGCGGAAGTGA 1360 2516_F AACACCTTA 2574_R AATCGT 2314 MUPR_X75439_2547_ TACGATTTCACTTCCGCAGCCA 188 MUPR_X75439_2605_ TCGTCCTCTCGAATCTCCGAT 1103 2572_F GATT 2630_R ATACC 2315 MUPR_X75439_2666_ TGCGTACAATACGCTTTATGAA 513 MUPR_X75439_2711_ TCAGATATAAATGGAACAAAT 981 2696_F ATTTTAACA 2740_R GGAGCCACT 2316 MUPR_X75439_2813_ TAATCAAGCATTGGAAGATGAA 165 MUPR_X75439_2867_ TCTGCATTTTTGCGAGCCTGT 1127 2843_F ATGCATACC 2890_R CTA 2317 MUPR_X75439_884_ TGACATGGACTCCCCCTATATA 447 MUPR_X75439_977_1007_ TGTACAATAAGGAGTCACCTT 1317 914_F ACTCTTGAG R ATGTCCCTTA 2318 CTXA_NC002505- TGGTCTTATGCCAAGAGGACAG 608 CTXA_NC002505- TCGTGCCTAACAAATCCCGTC 1109 1568114- AGTGAGT 1568114- TGAGTTC 1567341_114_142_F 1567341_194_221_R 2319 CTXA_NC002505- TCTTATGCCAAGAGGACAGAGT 411 CTXA_NC002505- TCGTGCCTAACAAATCCCGTC 1109 1568114- GAGTACT 1568114- TGAGTTC 1567341_117_145_F 1567341_194_221_R 2320 CTXA_NC002505- TGGTCTTATGCCAAGAGGACAG 608 CTXA_NC002505- TAACAAATCCCGTCTGAGTTC 855 1568114- AGTGAGT 1568114- CTCTTGCA 1567341_114_142_F 1567341_186_214_R 2321 CTXA_NC002505- TCTTATGCCAAGAGGACAGAGT 411 CTXA_NC002505- TAACAAATCCCGTCTGAGTTC 855 1568114- GAGTACT 1568114- CTCTTGCA 1567341_117_145_F 1567341_186_214_R 2322 CTXA_NC002505- AGGACAGAGTGAGTACTTTGAC 27 CTXA_NC002505- TCCCGTCTGAGTTCCTCTTGC 1027 1568114- CGAGGT 1568114- ATGATCA 1567341_129_156_F 1567341_180_207_R 2323 CTXA_NC002505- TGCCAAGAGGACAGAGTGAGTA 500 CTXA_NC002505- TAACAAATCCCGTCTGAGTTC 855 1568114- CTTTGA 1568114- CTCTTGCA 1567341_122_149_F 1567341_186_214_R 2324 INV_U22457-74- TGCTTATTTACCTGCACTCCCA 530 INV_U22457-74- TGACCCAAAGCTGAAAGCTTT 1154 3772_831_858_F CAACTG 3772_942_966_R ACTG 2325 INV_U22457-74- TGAATGCTTATTTACCTGCACT 438 INV_U22457-74- TAACTGACCCAAAGCTGAAAG 864 3772_827_857_F CCCACAACT 3772_942_970_R CTTTACTG 2326 INV_U22457-74- TGCTGGTAACAGAGCCTTATAG 526 INV_U22457-74- TGGGTTGCGTTGCAGATTATC 1296 3772_1555_1581_F GCGCA 3772_1619_1647_R TTTACCAA 2327 INV_U22457-74- TGGTAACAGAGCCTTATAGGCG 598 INV_U22457-74- TCATAAGGGTTGCGTTGCAGA 987 3772_1558_1585_F CATATG 3772_1622_1652_R TTATCTTTAC 2328 ASD_NC006570- TGAGGGTTTTATGCTTAAAGTT 459 ASD_NC006570-439714- TGATTCGATCATACGAGACAT 1188 439714- GGTTTTATTGGTT 438608_54_84_R TAAAACTGAG 438608_3_37_F 2329 ASD_NC006570- TAAAGTTGGTTTTATTGGTTGG 149 ASD_NC006570_439714- TCAAAATCTTTTGATTCGATC 948 439714- CGCGGA 438608_66_95_R ATACGAGAC 438608_18_45_F 2330 ASD_NC006570- TTAAAGTTGGTTTTATTGGTTG 647 ASD_NC006570_439714- TCCCAATCTTTTGATTCGATC 1016 439714- GCGCGGA 438608_67_95_R ATACGAGA 438608_17_45_F 2331 ASD_NC006570- TTTTATGCTTAAAGTTGGTTTT 709 ASD_NC006570-439714- TCTGCCTGAGATGTCGAAAAA 1128 439714- ATTGGTTGGC 438608_107_134_R AACGTTG 438608_9_40_F 2332 GALE_AF513299_171_ TCAGCTAGACCTTTTAGGTAAA 280 GALE_AF513299_241_ TCTCACCTACAGCTTTAAAGC 1122 200_F GCTAAGCT 271_R CAGCAAAATG 2333 GALE_AF513299_168_ TTATCAGCTAGACCTTTTAGGT 658 GALE_AF513299_245_ TCTCACCTACAGCTTTAAAGC 1121 199_F AAAGCTAAGC 271_R CAGCAA 2334 GALE_AF513299_168_ TTATCAGCTAGACCTTTTAGGT 658 GALE_AF513299_233_ TACAGCTTTAAAGCCAGCAAA 883 199_F AAAGCTAAGC 264_R ATGAATTACAG 2335 GALE_AF513299_169_ TCCCAGCTAGACCTTTTAGGTA 319 GALE_AF513299_252_ TTCAACACTCTCACCTACAGC 1374 198_F AAGCTAAG 279_R TTTAAAG 2336 PLA_AF053945_7371_ TTGAGAAGACATCCGGCTCACG 680 PLA_AF053945_7434_ TACGTATGTAAATTCCGCAAA 900 7403_F TTATTATGGTA 7468_R GACTTTGGCATTAG 2337 PLA_AF053945_7377_ TGACATCCGGCTCACGTTATTA 443 PLA_AF053945_7428_ TCCGCAAAGACTTTGGCATTA 1035 7403_F TGGTA 7455_R GGTGTGA 2338 PLA_AF053945_7377_ TGACATCCGGCTCACGTTATTA 444 PLA_AF053945_7430_ TAAATTCCGCAAAGACTTTGG 854 7404_F TGGTAC 7460_R CATTAGGTGT 2339 CAF_AF053947_33412_ TCCGTTATCGCCATTGCATTAT 329 CAF_AF053947_33498_ TAAGAGTGATGCGGGCTGGTT 866 33441_F TTGGAACT 33523_R CAACA 2340 CAF_AF053947_33426_ TGCATTATTTGGAACTATTGCA 499 CAF_AF053947_33483_ TGGTTCAACAAGAGTTGCCGT 1308 33458_F ACTGCTAATGC 33507_R TGCA 2341 CAF_AF053947_33407_ TCAGTTCCGTTATCGCCATTGC 291 CAF_AF053947_33483_ TTCAACAAGAGTTGCCGTTGC 1373 33429_F A 33504_R A 2342 CAF_AF053947_33407_ TCAGTTCCGTTATCGCCATTGC 293 CAF_AF053947_33494_ TGATGCGGGCTGGTTCAACAA 1184 33431_F ATT 33517_R GAG 2344 GAPA_NC_002505_1_ TCAATGAACGATCAACAAGTGA 260 GAPA_NC_002505_29_58_ TCCTTTATGCAACTTGGTATC 1060 28_F_1 TTGATG R_1 AACAGGAAT 2472 OMPA_NC000117_68_ TGCCTGTAGGGAATCCTGCTGA 507 OMPA_NC000117_145_ TCACACCAAGTAGTGCAAGGA 967 89_F 167_R TC 2473 OMPA_NC000117_798_ TGATTACCATGAGTGGCAAGCA 475 OMPA_NC000117_865_ TCAAAACTTGCTCTAGACCAT 947 821_F AG 893_R TTAACTCC 2474 OMPA_NC000117_645_ TGCTCAATCTAAACCTAAAGTC 521 OMPA_NC000117_757_ TGTCGCAGCATCTGTTCCTGC 1328 671_F GAAGA 777_R 2475 OMPA_NC000117_947_ TAACTGCATGGAACCCTTCTTT 157 OMPA_NC000117_1011_ TGACAGGACACAATCTGCATG 1153 973_F ACTAG 1040_R AAGTCTGAG 2476 OMPA_NC000117_774_ TACTGGAACAAAGTCTGCGACC 196 OMPA_NC000117_871_ TTCAAAAGTTGCTCGAGACCA 1371 795_F 894_R TTG 2477 OMPA_NC000117_457_ TTCTATCTCGTTGGTTTATTCG 676 OMPA_NC000117_511_ TAAAGAGACGTTTGGTAGTTC 851 483_F GAGTT 534_R ATTTGC 2478 OMPA_NC000117_687_ TAGCCCAGCACAATTTGTGATT 212 OMPA_NC000117_787_ TTGCCATTCATGGTATTTAAG 1406 710_F CA 816_R TGTAGCAGA 2479 OMPA_NC000117_540_ TGGCGTAGTAGAGCTATTTACA 571 OMPA_NC000117_649_ TTCTTGAACGCGAGGTTTCGA 1395 566_F GACAC 672_R TTG 2480 OMPA_NC000117_338_ TGCACGATGCGGAATGGTTCAC 492 OMPA_NC000117_417_ TCCTTTAAAATAACCGCTAGT 1058 360_F A 444_R AGCTCCT 2481 OMP2_NC000117_18_ TATGACCAAACTCATCAGACGA 234 OMP2_NC000117_71_91_ TCCCGCTGGCAAATAAACTCG 1025 40_F G R 2482 OMP2_NC000117_354_ TGCTACGGTAGGATCTCCTTAT 516 OMP2_NC000117_445_ TGGATCACTGCTTACGAACTC 1270 382_F CCTATTG 471_R AGCTTC 2483 OMP2_NC000117_1297_ TGGAAAGGTGTTGCAGCTACTC 537 OMP2_NC000117_1396_ TACGTTTGTATCTTCTGCAGA 903 1319_F A 1419_R ACC 2484 OMP2_NC000117_1465_ TCTGGTCCAACAAAAGGAACGA 407 OMP2_NC000117_1541_ TCCTTTCAATGTTACAGAAAA 1062 1493_F TTACAGG 1569_R CTCTACAG 2485 OMP2_NC000117_44_66 TGACGATCTTCGCGGTGACTAG 450 OMP2_NC000117_120_ TGTCAGCTAAGCTAATAACGT 1323 F T 148_R TTGTAGAG 2486 OMP2_NC000117_166_ TGACAGCGAAGAAGGTTAGACT 441 OMP2_NC000117_240_ TTGACATCGTCCCTCTTCACA 1396 190_F TGTCC 261_R G 2487 GYRA_NC000117_514_ TCAGGCATTGCGGTTGGGATGG 287 GYRA_NC000117_640_ TGCTGTAGGGAAATCAGGGCC 1251 536_F C 660_R 2488 GYRA_NC000117_801_ TGTGAATAAATCACGATTGATT 636 GYRA_NC000117_871_ TTGTCAGACTCATCGCGAACA 1419 827_F GAGCA 893_R TC 2489 GYRA_NC002952_219_ TGTCATGGGTAAATATCACCCT 632 GYRA_NC002952_319_ TCCATCCATAGAACCAAAGTT 1010 242_F CA 345_R ACCTTG 2490 GYRA_NC002952_964_ TACAAGCACTCCCAGCTGCA 176 GYRA_NC002952_1024 TCGCAGCGTGCGTGGCAC 1073 983_F 1041_R 2491 GYRA_NC002952_1505_ TCGCCCGCGAGGACGT 366 GYRA_NC002952_1546 TTGGTGCGCTTGGCGTA 1416 1520_F 1562_R 2492 GYRA_NC002952_59_ TCAGCTACATCGACTATGCGAT 279 GYRA_NC002952_124_ TGGCGATGCACTGGCTTGAG 1279 81_F G 143_R 2493 GYRA_NC002952_216_ TGACGTCATCGGTAAGTACCAC 452 GYRA_NC002952_313_ TCCGAAGTTGCCCTGGCCGTC 1032 239_F CC 333_R 2494 GYRA_NC002952_219_ TGTACTCGGTAAGTATCACCCG 625 GYRA_NC002952_308_ TAAGTTACCTTGCCCGTCAAC 873 242_2_F CA 330_R CA 2495 GYRA_NC002952_115_ TGAGATGGATTTAAACCTGTTC 453 GYRA_NC002952_220_ TGCGGGTGATACTTACCGAGT 1236 141_F ACCGC 242_R AC 2496 GYRA_NC002952_517_ TCAGGCATTGCGGTTGGGATGG 287 GYRA_NC002952_643_ TGCTGTAGGGAAATCAGGGCC 1251 539_F C 663_R 2497 GYRA_NC002952_273_ TCGTATGGCTCAATGGTGGAG 380 GYRA_NC002952_338_ TGCGGCAGCACTATCACCATC 1234 293_F 360_R CA 2498 GYRA_NC000912_257_ TGAGTAAGTTCCACCCGCACGG 462 GYRA_NC000912_346_ TCGAGCCGAAGTTACCCTGTC 1067 278_F 370_R CGTC 2504 ARCC_NC003923- TAGTpGATpAGAACpTpGTAGG 229 ARCC_NC003923- TCpTpTpTpCpGTATAAAAAG 1116 2725050- CpACpAATpCpGT 2725050- GACpCpAATpTpGG 2724595_135_161P_F 2724595_214_239P_R 2505 PTA_NC003923- TCTTGTpTpTpATGCpTpGGTA 417 PTA_NC003923-628885- TACpACpCpTGGTpTpTpCpG 904 628885- AAGCAGATGG 629355_314_342P_R TpTpTpTpGATGATpTpTpGT 629355_237_263P_F A 2517 CJMLST_ST1_1852_ TTTGCGGATGAAGTAGGTGCCT 708 CJMLST_ST1_1945_1977_ TGTTTTATGTGTAGTTGAGCT 1355 1883_F ATCTTTTTGC R TACTACATGAGC 2518 CJMLST_ST1_2963_ TGAAATTGCTACAGGCCCTTTA 428 CJMLST_ST1_3073_3097_ TCCCCATCTCCGCAAAGACAA 1020 2992_F GGACAAGG R TAAA 2519 CJMLST_ST1_2350_ TGCTTTTGATGGTGATGCAGAT 535 CJMLST_ST1_2447_2481_ TCTACAACACTTGATTGTAAT 1117 2378_F CGTTTGG R TTGCCTTGTTCTTT 2520 CJMLST_ST1_654_ TATGTCCAAGAAGCATAGCAAA 240 CJMLST_ST1_725_756_R TCGGAAACAAAGAATTCATTT 1084 684_F AAAAGCAAT TCTGGTCCAAA 2521 CJMLST_ST1_360_ TCCTGTTATTCCTGAAGTAGTT 347 CJMLST_ST1_454_487_R TGCTATATGCTACAACTGGTT 1245 395_F AATCAAGTTTGTTA CAAAAACATTAAG 2522 CJMLST_ST1_1231_ TGGCAGTTTTACAAGGTGCTGT 564 CJMLST_ST1_1312_1340_ TTTAGCTACTATTCTAGCTGC 1427 1258_F TTCATC R CATTTCCA 2523 CJMLST_ST1_3543_ TGCTGTAGCTTATCGCGAAATG 529 CJMLST_ST1_3656_3685_ TCAAAGAACCAGCACCTAATT 950 3574_F TCTTTGATTT R CATCATTTA 2524 CJMLST_ST1_1_17_F TAAAACTTTTGCCGTAATGATG 145 CJMLST_ST1_55_84_R TGTTCCAATAGCAGTTCCGCC 1348 GGTGAAGATAT CAAATTGAT 2525 CJMLST_ST1_1312_ TGGAAATGGCAGCTAGAATAGT 538 CJMLST_ST1_1383_1417_ TTTCCCCGATCTAAATTTGGA 1432 1342_F AGCTAAAAT R TAAGCCATAGGAAA 2526 CJMLST_ST1_2254_ TGGGCCTAATGGGCTTAATATC 582 CJMLST_ST1_2352_2379_ TCCAAACGATCTGCATCACCA 996 2286_F AATGAAAATTG R TCAAAAG 2527 CJMLST_ST1_1380_ TGCTTTCCTATGGCTTATCCAA 534 CJMLST_ST1_1486_1520_ TGCATGAAGCATAAAAACTGT 1205 1411_F ATTTAGATCG R ATCAAGTGCTTTTA 2528 CJMLST_ST1_3413_ TTGTAAATGCCGGTGCTTCAGA 692 CJMLST_ST1_3511_3542_ TGCTTGCTCAAATCATCATAA 1257 3437_F TCC R ACAATTAAAGC 2529 CJMLST_ST1_1130_ TACGCGTCTTGAAGCGTTTCGT 189 CJMLST_ST1_1203_1230_ TAGGATGAGCATTATCAGGGA 920 1156_F TATGA R AAGAATC 2530 CJMLST_ST1_2840_ TGGGGCTTTGCTTTATAGTTTT 591 CJMLST_ST1_2940_2973_ TAGCGATTTCTACTCCTAGAG 917 2872_F TTACATTTAAG R TTGAAATTTCAGG 2531 CJMLST_ST1_2058_ TATTCAAGGTGGTCCTTTGATG 241 CJMLST_ST1_2131_2162_ TTGGTTCTTACTTGTTTTGCA 1417 2084_F CATGT R TAAACTTTCCA 2532 CJMLST_ST1_553_ TCCTGATGCTCAAAGTGCTTTT 344 CJMLST_ST1_655_685_R TATTGCTTTTTTTGCTATGCT 942 585_F TTAGATCCTTT TCTTGGACAT 2564 GLTA_NC002163- TCATGTTGAGCTTAAACCTATA 299 GLTA_NC002163- TTTTGCTCATGATCTGCATGA 1443 1604930- GAAGTAAAAGC 1604930- AGCATAAA 1604529_306_338_F 1604529_352_380_R 2565 UNCA_NC002163- TCCCCCACGCTTTAATTGTTTA 322 UNCA_NC002163- TCGACCTGGAGGACGACGTAA 1065 112166- TGATGATTTGAG 112166- AATCA 112647_80_113_F 112647_146_171_R 2566 UNCA_NC002163- TAATGATGAATTAGGTGCGGGT 170 UNCA_NC002163- TGGGATAACATTGGTTGGAAT 1285 112166- TCTTT 112166- ATAAGCAGAAACATC 112647_233_259_F 112647_294_329_R 2567 PGM_NC002163- TCTTGATACTTGTAATGTGGGC 414 PGM_NC002163-327773- TCCATCGCCAGTTTTTGCATA 1012 327773- GATAAATATGT 328270_365_396_R ATCGCTAAAAA 2568 TKT_NC002163- TTATGAAGCGTGTTCTTTAGCA 661 TKT_NC002163- TCAAAACGCATTTTTACATCT 946 1569415- GGACTTCA 1569415- TCGTTAAAGGCTA 1569873_255_284_F 1569873_350_383_R 2570 GLTA_NC002163- TCGTCTTTTTGATTCTTTCCCT 381 GLTA_NC002163- TGTTCATGTTTAAATGATCAG 1347 1604930- GATAATGC 1604930- GATAAAAAGCACT 1604529_39_68_F 1604529_109_142_R 2571 TKT_NC002163- TGATCTTAAAAATTTCCGCCAA 472 TKT_NC002163- TGCCATAGCAAAGCCTACAGC 1214 1569415- CTTCATTC 1569415- ATT 1569903_33_62_F 1569903_139_162_R 2572 TKT_NC002163- TAAGGTTTATTGTCTTTGTGGA 164 TKT_NC002163- TACATCTCCTTCGATAGAAAT 886 1569415- GATGGGGATTT 1569415- TTCATTGCTATC 1569903_207_239_F 1569903_313_345_R 2573 TKT_NC002163- TAGCCTTTAACGAAAATGTAAA 213 TKT_NC002163- TAAGACAAGGTTTTGTGGATT 865 1569415- AATGCGTTTTGA 1569415- TTTTAGCTTGTT 1569903_350_383_F 1569903_449_481_R 2574 TKT_NC002163- TTCAAAAACTCCAGGCCATCCT 665 TKT_NC002163- TTGCCATAGCAAAGCCTACAG 1405 1569415- GAAATTTCAAC 1569415- CATT 1569903_60_92_F 1569903_139_163_R 2575 GLTA_NC002163- TCGTCTTTTTGATTCTTTCCCT 382 GLTA_NC002163- TGCCATTTCCATGTACTCTTC 1216 1604930- GATAATGCTC 1604930- TCTAACATT 1604529_39_70_F 1604529_139_168_R 2576 GLYA_NC002163- TCAGCTATTTTTCCAGGTATCC 281 GLYA_NC002163- ATTGCTTCTTACTTGCTTAGC 756 367572- AAGGTGG 367572- ATAAATTTTCCA 368079_386_414_F 368079_476_508_R 2577 GLYA_NC002163- TGGTGCGAGTGCTTATGCTCGT 611 GLYA_NC002163- TGCTCACCTGCTACAACAAGT 1246 367572- ATTAT 367572- CCAGCAAT 368079_148_174_F 368079_242_270_R 2578 GLYA_NC002163- TGTAAGCTCTACAACCCACAAA 622 GLYA_NC002163- TTCCACCTTGGATACCTGGAA 1381 367572- ACCTTACG 367572- AAATAGCTGAAT 368079_298_327_F 368079_384_416_R 2579 GLYA_NC002163- TGGTGGACATTTAACACATGGT 614 GLYA_NC002163- TCAAGCTCTACACCATAAAAA 961 367572- GCAAA 367572- AAGCTCTCA 368079_1_27_F 368079_52_81_R 2580 PGM_NC002163- TGAGCAATGGGGCTTTGAAAGA 455 PGM_NC002163-327746- TTTGCTCTCCGCCAAAGTTTC 1438 327746- 328270_356_379_R 328270_254_285_F 2581 PGM_NC002163- TGAAAAGGGTGAAGTAGCAAAT 425 PGM_NC002163-327746- TGCCCCATTGCTCATGATAGT 1219 327746- GGAGATAG 328270_241_267_R AGCTAC 328270_153_182_F 2582 PGM_NC002163- TGGCCTAATGGGCTTAATATCA 568 PGM_NC002163-327746- TGCACGCAAACGCTTTACTTC 1200 327746- ATGAAAATTG 328270_79_102_R AGC 328270_19_50_F 2583 UNCA_NC002163- TAAGCATGCTGTGGCTTATCGT 160 UNCA_NC002163- TGCCCTTTCTAAAAGTCTTGA 1220 112166- GAAATG 112166- GTGAAGATA 112647_114_141_F 112647_196_225_R 2584 UNCA_NC002163- TGCTTCGGATCCAGCAGCACTT 532 UNCA_NC002163- TGCATGCTTACTCAAATCATC 1206 112166- CAATA 112166- ATAAACAATTAAAGC 112647_3_29_F 112647_88_123_R 2585 ASPA_NC002163- TTAATTTGCCAAAAATGCAACC 652 ASPA_NC002163-96692- TGCAAAAGTAACGGTTACATC 1192 96692- AGGTAG 97166_403_432_R TGCTCCAAT 97166_308_335_F 2586 ASPA_NC002163- TCGCGTTGCAACAAAACTTTCT 370 ASPA_NC002163-96692- TCATGATAGAACTACCTGGTT 991 96692- AAAGTATGT 97166_316_346_R GCATTTTTGG 97166_228_258_F 2587 GLNA_NC002163- TGGAATGATGATAAAGATTTCG 547 GLNA_NC002163- TGAGTTTGAACCATTTCAGAG 1176 658085- CAGATAGCTA 658085- CGAATATCTAC 657609_244_275_F 657609_340_371_R 2588 TKT_NC002163- TCGCTACAGGCCCTTTAGGACA 371 TKT_NC002163- TCCCCATCTCCGCAAAGACAA 1020 1569415- AG 1569415- TAAA 1569903_107_130_F 1569903_212_236_R 2589 TKT_NC002163- TGTTCTTTAGCAGGACTTCACA 642 TKT_NC002163- TCCTTGTGCTTCAAAACGCAT 1057 1569415- AACTTGATAA 1569415- TTTTACATTTTC 1569903_265_296_F 1569903_361_393_R 2590 GLYA_NC002163- TGCCTATCTTTTTGCTGATATA 505 GLYA_NC002163- TCCTCTTGGGCCACGCAAAGT 1047 367572- GCACATATTGC 367572- TTT 368095_214246_F 368095_317_340_R 2591 GLYA_NC002163- TCCTTTGATGCATGTAATTGCT 353 GLYA_NC002163- TCTTGAGCATTGGTTCTTACT 1141 367572- GCAAAAGC 367572- TGTTTTGCATA 368095_415_444_F 368095_485_516_R 2592 PGM NC002163_21_ TCCTAATGGACTTAATATCAAT 332 PGM_NC002163_116_142_ TCAAACGATCCGCATCACCAT 949 54_F GAAAATTGTGGA R CAAAAG 2593 PGM_NC002163_149_ TAGATGAAAAAGGCGAAGTGGC 207 PGM_NC002163_247_277_ TCCCCTTTAAAGCACCATTAC 1023 176_F TAATGG R TCATTATAGT 2594 GLNA_NC002163- TGTCCAAGAAGCATAGCAAAAA 633 GLNA_NC002163- TCAAAAACAAAGAATTCATTT 945 658085- AAGCAA 658085- TCTGGTCCAAA 657609_79_106_F 657609_148_179_R 2595 ASPA_NC002163- TCCTGTTATTCCTGAAGTAGTT 347 ASPA_NC002163-96685- TCAAGCTATATGCTACAACTG 960 96685- AATCAAGTTTGTTA 97196_467_497_R GTTCAAAAAC 97196_367_402_F 2596 ASPA_NC002163- TGCCGTAATGATAGGTGAAGAT 502 ASPA_NC002163-96685- TACAACCTTCGGATAATCAGG 880 96685- ATACAAAGAGT 97196_95_127_R ATGAGAATTAAT 97196_1_33_F 2597 ASPA_NC002163- TGGAACAGGAATTAATTCTCAT 540 ASPA_NC002163-96685- TAAGCTCCCGTATCTTGAGTC 872 96685- CCTGATTATCC 97196_185_210_R GCCTC 97196_85_117_F 2598 PGM_NC002163- TGGCAGCTAGAATAGTAGCTAA 563 PGM_NC002163-327746- TCACGATCTAAATTTGGATAA 975 327746- AATCCCTAC 328270_230_261_R GCCATAGGAAA 328270_165_195_F 2599 PGM_NC002163- TGGGTCGTGGTTTTACAGAAAA 593 PGM_NC002163-327746- TTTTGCTCATGATCTGCATGA 1443 327746- TTTCTTATATATG 328270_353_381_R AGCATAAA 328270_252_286_F 2600 PGM_NC002163- TGGGATGAAAAAGCGTTCTTTT 577 PGM_NC002163-327746- TGATAAAAAGCACTAAGCGAT 1178 327746- ATCCATGA 328270_95_132_R GAAACAGC 328270_1_30_F 2601 PGM_NC002163- TAAACACGGCTTTCCTATGGCT 146 PGM_NC002163-327746- TCAAGTGCTTTTACTTCTATA 963 327746- TATCCAAAT 328270_314_345_R GGTTTAAGCTC 328270_220_250_F 2602 UNCA_NC002163- TGTAGCTTATCGCGAAATGTCT 628 UNCA_NC002163- TGCTTGCTCTTTCAAGCAGTC 1258 112166- TTGATTTT 112166- TTGAATGAAG 112647_123_152_F 112647_199_229_R 2603 UNCA_NC002163- TCCAGATGGACAAATTTTCTTA 313 UNCA_NC002163- TCCGAAACTTGTTTTGTAGCT 1031 112166- GAAACTGATTT 112166- TTAATTTGAGC 112647_333_365_F 112647_430_461_R 2734 GYRA_AY291534_237_ TCACCCTCATGGTGATTCAGCT 265 GYRA_AY291534_268_ TTGCGCCATACGTACCATCGT 1407 264_F GTTTAT 288_R 2735 GYRA_AY291534_224_ TAATCGGTAAGTATCACCCTCA 167 GYRA_AY291534_256_ TGCCATACGTACCATCGTTTC 1213 252_F TGGTGAT 285_R ATAAACAGC 2736 GYRA_AY291534_170_ TAGGAATTACGGCTGATAAAGC 221 GYRA_AY291534_268_ TTGCGCCATACGTACCATCGT 1407 198_F GTATAAA 288_R 2737 GYRA_AY291534_224_ TAATCGGTAAGTATCACCCTCA 167 GYRA_AY291534_319_ TATCGACAGATCCAAAGTTAC 935 252_F TGGTGAT 346_R CATGCCC 2738 GYRA_NC002953- TAAGGTATGACACCGGATAAAT 163 GYRA_NC002953-7005- TCTTGAGCCATACGTACCATT 1142 7005- CATATAAA 9668_265_287_R GC 9668_166_195_F 2739 GYRA_NC002953- TAATGGGTAAATATCACCCTCA 171 GYRA_NC002953-7005- TATCCATTGAACCAAAGTTAC 933 7005- TGGTGAC 9668_316_343_R CTTGGCC 9668_221_249_F 2740 GYRA_NC002953- TAATGGGTAAATATCACCCTCA 171 GYRA_NC002953-7005- TAGCCATACGTACCATTGCTT 912 7005- TGGTGAC 9668_253_283_R CATAAATAGA 9668_221_249_F 2741 GYRA_NC002953- TCACCCTCATGGTGACTCATCT 264 GYRA_NC002953-7005- TCTTGAGCCATACGTACCATT 1142 7005- ATTTAT 9668_265_287_R GC 9668_234_261_F 2842 CAPC_AF188935- TGGGATTATTGTTATCCTGTTA 578 CAPC_AF188935-56074- TGGTAACCCTTGTCTTTGAAT 1299 56074- TGCCATTTGAGA 55628_348_378_R TGTATTTGCA 55628_271_304_F 2843 CAPC_AF188935- TGATTATTGTTATCCTGTTATG 476 CAPC_AF188935-560740 TGTAACCCTTGTCTTTGAATp 1314 56074- CpCpATpTpTpGAG 55628_349_377P_R TpGTATpTpTpGC 55628_273_303P_F 2844 CAPC_AF188935- TCCGTTGATTATTGTTATCCTG 331 CAPC_AF188935-56074- TGTTAATGGTAACCCTTGTCT 1344 56074- TTATGCCATTTGAG 55628_349_384_R TTGAATTGTATTTGC 55628_268_303_F 2845 CAPC_AF188935- TCCGTTGATTATTGTTATCCTG 331 CAPC_AF188935-56074- TAACCCTTGTCTTTGAATTGT 860 56074- TTATGCCATTTGAG 55628_337_375_R ATTTGCAATTAATCCTGG 55628_268_303_F 2846 PARC_X95819_33_58_F TCCAAAAAAATCAGCGCGTACA 302 PARC_X95819_121_153_R TAAAGGATAGCGGTAACTAAA 852 GTGG TGGCTGAGCCAT 2847 PARC_X95819_65_92_F TACTTGGTAAATACCACCCACA 199 PARC_X95819_157_178_R TACCCCAGTTCCCCTGACCTT 889 TGGTGA C 2848 PARC_X95819_69_93_F TGGTAAATACCACCCACATGGT 596 PARC_X95819_97_128_R TGAGCCATGAGTACCATGGCT 1169 GAC TCATAACATGC 2849 PARC_NC003997- TTCCGTAAGTCGGCTAAAACAG 668 PARC_NC003997- TCCAAGTTTGACTTAAACGTA 1001 3362578- TCG 3362578- CCATCGC 3365001_181_205_F 3365001_256_283_R 2850 PARC_NC003997- TGTAACTATCACCCGCACGGTG 621 PARC_NC003997- TCGTCAACACTACCATTATTA 1099 3362578- AT 3362578- CCATGCATCTC 3365001_217_240_F 3365001_304_335_R 2851 PARC_NC003997- TGTAACTATCACCCGCACGGTG 621 PARC_NC003997- TGACTTAAACGTACCATCGCT 1162 3362578- AT 3362578- TCATATACAGA 3365001_217_240_F 3365001_244_275_R 2852 GYRA_AY642140_−1_ TAAATCTGCCCGTGTCGTTGGT 150 GYRA_AY642140_71_100_ TGCTAAAGTCTTGAGCCATAC 1242 24_F GAC R GAACAATGG 2853 GYRA_AY642140_26_ TAATCGGTAAATATCACCCGCA 166 GYRA_AY642140_121_ TCGATCGAACCGAAGTTACCC 1069 54_F TGGTGAC 146_R TGACC 2854 GYRA_AY642140_26_ TAATCGGTAAATATCACCCGCA 166 GYRA_AY642140_58_89_R TGAGCCATACGAACAATGGTT 1168 54_F TGGTGAC TCATAAACAGC 2860 CYA_AF065404_1348_ TCCAACGAAGTACAATACAAGA 305 CYA_AF065404_1448_ TCAGCTGTTAACGGCTTCAAG 983 1379_F CAAAAGAAGG 1472_R ACCC 2861 LEF_BA_AF065404_ TCGAAAGCTTTTGCATATTATA 354 LEF_BA_AF065404_843_ TCTTTAAGTTCTTCCAAGGAT 1144 751_781_F TCGAGCCAC 881_R AGATTTATTTCTTGTTCG 2862 LEF_BA_AF065404_ TGCATATTATATCGAGCCACAG 498 LEF_BA_AF065404_843_ TCTTTAAGTTCTTCCAAGGAT 1144 762_788_F CATCG 881_R AGATTTATTTCTTGTTCG 2917 MUTS_AY698802_106_ TCCGCTGAATCTGTCGCCGC 326 MUTS_AY698802_172_ TGCGGTCTGGCGCATATAGGT 1237 125_F 193_R A 2918 MUTS_AY698802_172_ TACCTATATGCGCCAGACCGC 187 MUTS_AY698802_228_ TCAATCTCGACTTTTTGTGCC 965 192_F 252_R GGTA 2919 MUTS_AY698802_228_ TACCGGCGCAAAAAGTCGAGAT 186 MUTS_AY698802_314_ TCGGTTTCAGTCATCTCCACC 1097 252_F TGG 342_R ATAAAGGT 2920 MUTS_AY698802_315_ TCTTTATGGTGGAGATGACTGA 419 MUTS_AY698802_413_ TGCCAGCGACAGACCATCGTA 1210 342_F AACCGA 433_R 2921 MUTS_AY698802_394_ TGGGCGTGGAACGTCCAC 585 MUTS_AY698802_497_ TCCGGTAACTGGGTCAGCTCG 1040 411_F 519_R AA 2922 AB_MLST-11- TGGGcGATGCTGCgAAATGGTT 583 AB_MLST-11- TAGTATCACCACGTACACCCG 923 OIF007_991_1018_F AAAAGA OIF007_1110_1137_R GATCAGT 2927 GAPA_NC002505_694_ TCAATGAACGACCAACAAGTGA 259 GAPA_NC_002505_29_58_ TCCTTTATGCAACTTGGTATC 1060 721_F TTGATG R_1 AACAGGAAT 2928 GAPA_NC002505_694_ TCGATGAACGACCAACAAGTGA 361 GAPA_NC002505_769_ TCCTTTATGCAACTTGGTATC 1061 721_2_F TTGATG 798_2_R AACCGGAAT 2929 GAPA_NC002505_694_ TCGATGAACGACCAACAAGTGA 361 GAPA_NC002505_769_ TCCTTTATGCAACTTAGTATC 1059 721_2_F TTGATG 798_3_R AACCGGAAT 2932 INFB_EC_1364_1394_F TTGCTCGTGGTGCACAAGTAAC 688 INFB_EC_1439_1468_R TTGCTGCTTTCGCATGGTTAA 1410 GGATATTAC TCGCTTCAA 2933 INFB_EC_1364_1394_ TTGCTCGTGGTGCAIAAGTAAC 689 INFB_EC_1439_1468_R TTGCTGCTTTCGCATGGTTAA 1410 2_F GGATATIAC TCGCTTCAA 2934 INFB_EC_80_110_F TTGCCCGCGGTGCGGAAGTAAC 685 INFB_EC_1439_1468_R TTGCTGCTTTCGCATGGTTAA 1410 CGATATTAC TCGCTTCAA 2949 ACS_NC002516- TCGGCGCCTGCCTGATGA 376 ACS_NC002516-970624- TGGACCACGCCGAAGAACGG 1265 970624- 971013_364_383_R 971013_299_316_F 2950 ARO_NC002516- TCACCGTGCCGTTCAAGGAAGA 267 ARO_NC002516-26883- TGTGTTGTCGCCGCGCAG 1341 26883- G 27380_111_128_R 27380_4_26_F 2951 ARO_NC002516- TTTCGAAGGGCCTTTCGACCTG 705 ARO_NC002516-26883- TCCTTGGCATACATCATGTCG 1056 26883- 27380_459_484_R TAGCA 27380_356_377_F 2952 GUA_NC002516- TGGACTCCTCGGTGGTCGC 551 GUA_NC002516- TCGGCGAACATGGCCATCAC 1091 4226546- 4226546- 4226174_23_41_F 4226174_127_146_R 2953 GUA_NC002516- TGACCAGGTGATGGCCATGTTC 448 GUA_NC002516- TGCTTCTCTTCCGGGTCGGC 1256 4226546- G 4226546- 4226174_120_142_F 4226174_214_233_R 2954 GUA_NC002516- TTTTGAAGGTGATCCGTGCCAA 710 GUA_NC002516- TGCTTGGTGGCTTCTTCGTCG 1259 4226546- CG 4226546- AA 4226174_155_178_F 4226174_265_287_R 2955 GUA_NC002516- TTCCTCGGCCGCCTGGC 670 GUA_NC002516- TGCGAGGAACTTCACGTCCTG 1229 4226546- 4226546- C 4226174_190_206_F 4226174_288_309_R 2956 GUA_NC002516- TCGGCCGCACCTTCATCGAAGT 374 GUA_NC002516- TCGTGGGCCTTGCCGGT 1111 4226546- 4226546- 4226174_242_263_F 4226174_355_371_R 2957 MUT_NC002516- TGGAAGTCATCAAGCGCCTGGC 545 MUT_NC002516- TCACGGGCCAGCTCGTCT 978 5551158- 5551158- 5550717_5_26_F 5550717_99_116_R 2958 MUT_NC002516- TCGAGCAGGCGCTGCCG 358 MUT_NC002516- TCACCATGCGCCCGTTCACAT 971 5551158- 5551158- A 5550717_152_168_F 5550717_256_277_R 2959 NUO_NC002516- TCAACCTCGGCCCGAACCA 249 NUO_NC002516- TCGGTGGTGGTAGCCGATCTC 1095 2984589- 2984589- 2984954_8_26_F 2984954_97_117_R 2960 NUO_NC002516- TACTCTCGGTGGAGAAGCTCGC 195 NUO_NC002516- TTCAGGTACAGCAGGTGGTTC 1376 2984589- 29845899- AGGAT 2984954_218_239_F 2984954_301_326_R 2961 PPS_NC002516- TCCACGGTCATGGAGCGCTA 311 PPS_NC002516- TCCATTTCCGACACGTCGTTG 1014 1915014- 1915014- ATCAC 1915383_44_63_F 1915383_140_165_R 2962 PPS_NC002516- TCGCCATCGTCACCAACCG 365 PPS_NC002516- TCCTGGCCATCCTGCAGGAT 1052 1915014- 1915014- 1915383_240_258_F 1915383_341_360_R 2963 TRP_NC002516- TGCTGGTACGGGTCGAGGA 527 TRP_NC002516-671831- TCGATCTCCTTGGCGTCCGA 1071 671831- 672273_131_150_R 672273_24_42_F 2964 TRP_NC002516- TGCACATCGTGTCCAACGTCAC 490 TRP_NC002516-671831- TGATCTCCATGGCGCGGATCT 1182 671831- 672273_362_383_R T 672273_261_282_F 2972 AS_MLST-11- TGGGIGATGCTGCIAAATGGTT 592 AB_MLST-11- TAGTATCACCACGTACICCIG 924 OIF007_1007_1034_F AAAAGA OIF007_1126_1153_R GATCAGT 2993 OMPU_NC002505- TTCCCACCGATATCATGGCTTA 667 OMPU_NC002505_544_ TCGGTCAGCAAAACGGTAGCT 1094 674828- CCACGG 567_R TGC 675880_428_455_F 2994 GAPA_NC002505- TCCTCAATGAACGAICAACAAG 335 GAPA_NC002505- TTTTCCCTTTATGCAACTTAG 1442 506780- TGATTGATG 506780- TATCAACIGGAAT 507937_691_721_F 507937_769_802_R 2995 GAPA_NC002505- TCCTCIATGAACGAICAACAAG 339 GAPA_NC002505- TCCATACCTTTATGCAACTTI 1008 506780- TGATTGATG 506780- GTATCAACIGGAAT 507937_691_721_2_F 507937_769_803_R 2996 GAPA_NC002505- TCTCGATGAACGACCAACAAGT 396 GAPA_NC002505- TCGGAAATATTCTTTCAATAC 1085 506780- GATTGATG 506780- CTTTATGCAACT 507937_692_721_F 507937_785_817_R 2997 GAPA_NC002505- TCCTCGATGAACGAICAACAAG 337 GAPA_NC002505- TCGGAAATATTCTTTCAATAC 1085 506780- TIATTGATG 506780- CTTTATGCAACT 507937_691_721_3_F 507937_785_817_R 2998 GAPA_NC002505- TCCTCAATGAATGATCAACAAG 336 GAPA_NC002505- TCGGAAATATTCTTTCAATIC 1087 506780- TGATTGATG 506780- CTTTITGCAACTT 507937_691_721_4_F 507937_784_817_R 2999 GAPA_NC002505- TCCTCIATGAAIGAICAACAAG 340 GAPA_NC002505- TCGGAAATATTCTTTCAATAC 1086 506780- TIATTGATG 506780- CTTTATGCAACTT 507937_691_721_5_F 507937_784_817_2_R 3000 GAPA_NC002505- TCCTCGATGAATGAICAACAAG 338 GAPA_NC002505- TTTCAATACCTTTATGCAACT 1430 506780- TIATTGATG 506780- TIGTATCAACIGGAAT 507937_691_721_6_F 507937_769_805_R 3001 CTXB_NC002505- TCAGCATATGCACATGGAACAC 275 CTXB_NC002505- TCCCGGCTAGAGATTCTGTAT 1026 1566967- CTCA 1566967- ACGA 1567341_46_71_F 1567341_139_163_R 3002 CTXB_NC002505- TCAGCATATGCACATGGAACAC 274 CTXB_NC002505- TCCGGCTAGAGATTCTGTATA 1038 1566967- CTC 15669670 CGAAAATATC 1567341_46_70_F 1567341_132_162_R 3003 CTXB_NC002505- TCAGCATATGCACATGGAACAC 274 CTXB_NC002505- TGCCGTATACGAAAATATCTT 1225 1566967- CTC 1566967- ATCATTTAGCGT 1567341_46_70_F 1567341_118_150_R 3004 TUFB_NC002758- TACAGGCCGTGTTGAACGTGG 180 TUFB_NC002758- TCAGCGTAGTCTAATAATTTA 982 615038- 615038- CGGAACATTTC 616222_684_704_F 616222_778_809_R 3005 TUFB_NC002758- TGCCGTGTTGAACGTGGTCAAA 503 TUFB_NC002758- TGCTTCAGCGTAGTCTAATAA 1255 615038- T 615038- TTTACGGAAC 616222_688_710_F 616222_783_813_R 3006 TUFB_NC002758- TGTGGTCAAATCAAAGTTGGTG 638 TUFB_NC002758- TGCGTAGTCTAATAATTTACG 1238 615038- AAGAA 615038- GAACATTTC 616222_700_726_F 616222_778_807_R 3007 TUFB_NC002758- TGGTCAAATCAAAGTTGGTGAA 607 TUFB_NC002758- TGCGTAGTCTAATAATTTACG 1238 615038- GAA 615038- GAACATTTC 616222_702_726_F 616222_778_807_R 3008 TUFB_NC002758- TGAACGTGGTCAAATCAAAGTT 431 TUFB_NC002758- TCACCAGCTTCAGCGTAGTCT 970 615038- GGTGAAGAA 615038- AATAATTTACGGA 616222_696_726_F 616222_785_818_R 3009 TUFB_NC002758- TCGTGTTGAACGTGGTCAAATC 386 TUFB_NC002758- TCTTCAGCGTAGTCTAATAAT 1134 615038- AAAGT 615038- TTACGGAACATTTC 616222_690_716_F 616222_778_812_R 3010 MECI-R_NC003923- TCACATATCGTGAGCAATGAAC 261 MECI-R_NC003923- TGTGATATGGAGGTGTAGAAG 1332 41798- TG 41798-41609_89_112_R GTG 41609_36_59_F 3011 MECI-R_NC003923- TGGGCGTGAGCAATGAACTGAT 584 MECI-R_NC003923- TGGGATGGAGGTGTAGAAGGT 1287 41798- TATAC 41798-41609_81_110_R GTTATCATC 41609_40_66_F 3012 MECI-R_NC003923- TGGACACATATCGTGAGCAATG 549 MECI-R_NC003923- TGGGATGGAGGTGTAGAAGGT 1286 41798- AACTGA 41798-41609_81_110_R GTTATCATC 41609_33_60_2_F 3013 MECI-R_NC003923- TGGGTTTACACATATCGTGAGC 595 MECI-R_NC003923- TGGGGATATGGAGGTGTAGAA 1290 41798- AATGAACTGA 41798-41609_81_113_R GGTGTTATCATC 41609_29_60_F 3014 MUPR_X75439_2490_ TGGGCTCTTTCTCGCTTAAACA 587 MUPR_X75439_2548_ TCTGGCTGCGGAAGTGAAATC 1130 2514_F CCT 2570_R GT 3015 MUPR_X75439_2490_ TGGGCTCTTTCTCGCTTAAACA 586 MUPR_X75439_2547_ TGGCTGCGGAAGTGAAATCGT 1281 2513_F CC 2568R A 3016 MUPR_X75439_2482_ TAGATAATTGGGCTCTTTCTCG 205 MUPR_X75439_2551_ TAATCTGGCTGCGGAAGTGAA 876 2510_F CTTAAAC 2573_R AT 3017 MUPR_X75439_2490_ TGGGCTCTTTCTCGCTTAAACA 587 MUPR_X75439_2549_ TAATCTGGCTGCGGAAGTGAA 877 2514_F CCT 2573_R ATCG 3018 MUPR_X75439_2482_ TAGATAATTGGGCTCTTTCTCG 205 MUPR_X75439_2559_ TGGTATATTCGTTAATTAATC 1303 2510_F CTTAAAC 2589_R TGGCTGCGGA 3019 MUPR_X75439_2490_ TGGGCTCTTTCTCGCTTAAACA 587 MUPR_X75439_2554_ TCGTTAATTAATCTGGCTGCG 1112 2514_F CCT 2581_R GAAGTGA 3020 AROE_NC003923- TGATGGCAAGTGGATAGGGTAT 474 AROE_NC003923- TAAGCAATACCTTTACTTGCA 868 1674726- AATACAG 1674726- CCACCT 1674277_204_232_F 1674277_309_335_R 3021 AROE_NC003923- TGGCGAGTGGATAGGGTATAAT 570 AROE_NC003923- TTCATAAGCAATACCTTTACT 1378 1674726- ACAG 1674726- TGCACCAC 1674277_207_232_F 1674277_311_339_R 3022 AROE_NC003923- TGGCpAAGTpGGATpAGGGTpA 572 AROE_NC003923- TAAGCAATACCpTpTpTpACT 867 1674726- TpAATpACpAG 1674726- pTpGCpACpCpAC 1674277_207_232P_F 1674277_311_335P_R 3023 ARCC_NC003923- TCTGAAATGAATAGTGATAGAA 398 ARCC_NC003923- TCTTCTTCTTTCGTATAAAAA 1137 2725050- CTGTAGGCAC 2725050- GGACCAATTGG 2724595_124_155_F 2724595_214_245_R 3024 ARCC_NC003923- TGAATAGTGATAGAACTGTAGG 437 ARCC_NC003923- TCTTCTTTCGTATAAAAAGGA 1139 2725050- CACAATCGT 2725050- CCAATTGGTT 2724595_131_161_F 2724595_212_242_R 3025 ARCC_NC003923- TGAATAGTGATAGAACTGTAGG 437 ARCC_NC003923- TGCGCTAATTCTTCAACTTCT 1232 2725050- CACAATCGT 2725050- TCTTTCGT 2724595_131_161_F 2724595_232_260_R 3026 PTA_NC003923- TACAATGCTTGTTTATGCTGGT 177 PTA_NC003923-628885- TGTTCTTGATACACCTGGTTT 1350 628885- AAAGCAG 629355_322_351_R CGTTTTGAT 629355_231_259_F 3027 PTA_NC003923- TACAATGCTTGTTTATGCTGGT 177 PTA_NC003923-628885- TGGTACACCTGGTTTCGTTTT 1301 628885- AAAGCAG 629355_314_345_R GATGATTTGTA 629355_231_259_F 3028 PTA_NC003923- TCTTGTTTATGCTGGTAAAGCA 418 PTA_NC003923-628885- TGTTCTTGATACACCTGGTTT 1350 628885- GATGG 629355_322_351_R CGTTTTGAT 629355_237_263_F 3346 RPOB_NC000913_3704_ TGAACCACTTGGTTGACGACAA 1448 RPOB_NC000913_3793_ TCACCGAAACGCTGACCACCG 1461 3731_F GATGCA 3815_R AA 3347 RPOB_NC000913_3704_ TGAACCACTTGGTTGACGACAA 1448 RPOB_NC000913_3796_ TCCATCTCACCGAAACGCTGA 1464 3731_F GATGCA 3821_R CCACC 3348 RPOB_NC000913_3714_ TGTTGATGACAAGATGCACGCG 1451 RPOB_NC000913_3796_ TCCATCTCACCGAAACGCTGA 1464 3740_F CGTTC 3821_R CCACC 3349 RPOB_NC000913_3720_ TGACAAGATGCACGCGCGTTC 1450 RPOB_NC000913_3796_ TCTCACCGAAACGCTGACCAC 1463 3740_F 3817_R C 3350 RPLB_EC_690_710_F TCCACACGGTGGTGGTGAAGG 309 RPLB_NC000913_739_ TCCAAGCGCAGGTTTACCCCA 1458 762_R TGG 3351 RPLB_EC_690_710_F TCCACACGGTGGTGGTGAAGG 309 RPLB_NC000913_742_ TCCAAGCGCAGGTTTACCCCA 1460 762_R 3352 RPLB_NC000913_674_ TGAACCCTAATGATCACCCACA 1445 RPLB_NC000913_739_ TCCAAGCGCAGGTTTACCCCA 1458 698_F CGG 762_R TGG 3353 RPLB_NC000913_674_ TGAACCCTAACGATCACCCACA 1447 RPLB_NC000913_742_ TCCAAGCGCAGGTTTACCCCA 1460 698_2_F CGG 762_R 3354 RPLB_EC_690_710_F TCCACACGGTGGTGGTGAAGG 309 RPLB_NC000913_742_ TCCAAGCGCTGGTTTACCCCA 1459 762_2_R 3355 RPLB_NC000913_651_ TCCAACTGTTCGTGGTTCTGTA 1446 RPLB_NC000913_739_ TCCAAGCGCAGGTTTACCCCA 1458 680_F ATGAACCC 762_R TGG 3356 RPOB_NC000913_3789_ TCAGTTCGGTGGCCAGCGCTTC 1452 RPOB_NC000913_3868_ TACGTCGTCCGACTTGACCGT 1467 3812_F GG 3894_R CAGCAT 3357 RPOB_NC000913_3789_ TCAGTTCGGTGGCCAGCGCTTC 1452 RPOB_NC000913_3862_ TCCGACTTGACCGTCAGCATC 1465 3812_F GG 3887_R TCCTG 3358 RPOB_NC000913_3789_ TCAGTTCGGTGGTCAGCGCTTC 1453 RPOB_NC000913_3862_ TCGTCGGACTTGATGGTCAGC 1466 3812_2_F GG 3890_R AGCTCCTG 3359 RPOB_NC000913_3739_ TCCACCGGTCCGTACTCCATGA 1449 RPOB_NC000913_3794_ CCGAAGCGCTGGCCACCGA 1462 3761_F T 3812_R 3360 GYRB_NC002737_852_ TCATACTCATGAAGGTGGAACG 1444 GYRB_NC002737_973_ TGCAGTCAAGCCTTCACGAAC 1457 879_F CATGAA 996_R ATC 3361 TUFB_NC002758_275_ TGATCACTGGTGCTGCTCAAAT 1454 TUFB_NC002758_337_ TGGATGTGTTCACGAGTTTGA 1468 298_F GG 362_R GGCAT 3362 VALS_NC000913_1098_ TGGCGACCGTGGCGGCGT 1455 VALS_NC000913_1198_ TACTGCTTCGGGACGAACTGG 1469 1115_F 1226_R ATGTCGCC 3363 VALS_NC000913_1105_ TGTGGCGGCGTGGTTATCGAAC 1456 VALS_NC000913_1207_ TCGTACTGCTTCGGGACGAAC 1470 1127_F C 1229_R TG

Primer pair name codes and reference sequences are shown in Table 3. The primer name code typically represents the gene to which the given primer pair is targeted. The primer pair name may include specific coordinates with respect to a reference sequence defined by an extraction of a section of sequence or defined by a GenBank gi number, or the corresponding complementary sequence of the extraction, or the entire GenBank gi number as indicated by the label “no extraction.” Where “no extraction” is indicated for a reference sequence, the coordinates of a primer pair named to the reference sequence are with respect to the GenBank gi listing. Gene abbreviations are shown in bold type in the “Gene Name” column.

To determine the exact primer hybridization coordinates of a given pair of primers on a given bioagent nucleic acid sequence and to determine the sequences, molecular masses and base compositions of an amplification product to be obtained upon amplification of nucleic acid of a known bioagent with known sequence information in the region of interest with a given pair of primers, one with ordinary skill in bioinformatics is capable of obtaining alignments of the primers disclosed herein with the GenBank gi number of the relevant nucleic acid sequence of the known bioagent. For example, the reference sequence GenBank gi numbers (Table 3) provide the identities of the sequences which can be obtained from GenBank. Alignments can be done using a bioinformatics tool such as BLASTn provided to the public by NCBI (Bethesda, Md.). Alternatively, a relevant GenBank sequence may be downloaded and imported into custom programmed or commercially available bioinformatics programs wherein the alignment can be carried out to determine the primer hybridization coordinates and the sequences, molecular masses and base compositions of the amplification product. For example, to obtain the hybridization coordinates of primer pair number 2095 (SEQ ID NOs: 456:1261), First the forward primer (SEQ ID NO: 456) is subjected to a BLASTn search on the publicly available NCBI BLAST website. “RefSeq_Genomic” is chosen as the BLAST database since the gi numbers refer to genomic sequences. The BLAST query is then performed. Among the top results returned is a match to GenBank gi number 21281729 (Accession Number NC_(—)003923). The result shown below, indicates that the forward primer hybridizes to positions 1530282.1530307 of the genomic sequence of Staphylococcus aureus subsp. aureus MW2 (represented by gi number 21281729).

The hybridization coordinates of the reverse primer (SEQ ID NO: 1261) can be determined in a similar manner and thus, the bioagent identifying amplicon can be defined in terms of genomic coordinates. The query/subject arrangement of the result would be presented in Strand=Plus/Minus format because the reverse strand hybridizes to the reverse complement of the genomic sequence. The preceding sequence analyses are well known to one with ordinary skill in bioinformatics and thus, Table 3 contains sufficient information to determine the primer hybridization coordinates of any of the primers of Table 2 to the applicable reference sequences described therein.

TABLE 3 Primer Name Codes and Reference Sequences Reference GenBank Primer name gi code Gene Name Organism number 16S_EC 16S rRNA (16S ribosomal RNA Escherichia 16127994 gene) coli 23S_EC 23S rRNA (23S ribosomal RNA Escherichia 16127994 gene) coli CAPC_BA capC (capsule biosynthesis Bacillus 6470151 gene) anthracis CYA_BA cya (cyclic AMP gene) Bacillus 4894216 anthracis DNAK_EC dnaK (chaperone dnaK gene) Escherichia 16127994 coli GROL_EC groL (chaperonin groL) Escherichia 16127994 coli HFLB_EC hflb (cell division protein Escherichia 16127994 peptidase ftsH) coli INFB_EC infB (protein chain Escherichia 16127994 initiation factor infB gene) coli LEF_BA lef (lethal factor) Bacillus 21392688 anthracis PAG_BA pag (protective antigen) Bacillus 21392688 anthracis RPLB_EC rplB (50S ribosomal protein Escherichia 16127994 L2) coli RPLB_NC000913 rplB (50S ribosomal protein Escherichia 49175990 L2) coli RPOB_EC rpoB (DNA-directed RNA Escherichia 6127994 polymerase beta chain) coli RPOB_NC000913 rpoB (DNA-directed RNA Escherichia 49175990 polymerase beta chain) coli RPOC_EC rpoC (DNA-directed RNA Escherichia 16127994 polymerase beta' chain) coli SP101ET_SPET_11 Artificial Sequence Artificial 15674250 Concatenation comprising: Sequence* - gki (glucose kinase) partial gene gtr (glutamine transporter sequences of protein) Streptococcus murI (glutamate racemase) pyogenes mutS (DNA mismatch repair protein) xpt (xanthine phosphoribosyl transferase) yqiL (acetyl-CoA-acetyl transferase) tkt (transketolase) SSPE_BA sspE (small acid-soluble Bacillus 30253828 spore protein) anthracis TUFB_EC tufB (Elongation factor Tu) Escherichia 16127994 coli VALS_EC valS (Valyl-tRNA synthetase) Escherichia 16127994 coli VALS_NC000913 valS (Valyl-tRNA synthetase) Escherichia 49175990 coli ASPS_EC aspS (Aspartyl-tRNA Escherichia 16127994 synthetase) coli CAF1_AF053947 caf1 (capsular protein caf1) Yersinia 2996286 pestis INV_U22457 inv (invasin) Yersinia 1256565 pestis LL_NC003143 Y. pestis specific Yersinia 16120353 chromosomal genes - pestis difference region BONTA_X52066 BoNT/A (neurotoxin type A) Clostridium 40381 botulinum MECA_Y14051 mecA methicillin resistance Staphylococcus 2791983 gene aureus TRPE_AY094355 trpE (anthranilate synthase Acinetobacter 20853695 (large component)) baumanii RECA_AF251469 recA (recombinase A) Acinetobacter 9965210 baumanii GYRA_AF100557 gyrA (DNA gyrase subunit A) Acinetobacter 4240540 baumanii GYRB_AB008700 gyrB (DNA gyrase subunit B) Acinetobacter 4514436 baumanii GYRB_NC002737 gyrB (DNA gyrase subunit B) Streptococcus 15674250 pyogenes M1 GAS WAAA_Z96925 waaA (3-deoxy-D-manno- Acinetobacter 2765828 octulosonic-acid baumanii transferase) CJST_CJ Artificial Sequence Artificial 15791399 Concatenation comprising: Sequence* - tkt (transketolase) partial gene glyA (serine sequences of hydroxymethyltransferase) Campylobacter gltA (citrate synthase) jejuni aspA (aspartate ammonia lyase) glnA (glutamine synthase) pgm (phosphoglycerate mutase) uncA (ATP synthetase alpha chain) RNASEP_BDP RNase P (ribonuclease P) Bordetella 33591275 pertussis RNASEP_BKM RNase P (ribonuclease P) Burkholderia 53723370 mallei RNASEP_BS RNase P (ribonuclease P) Bacillus 16077068 subtilis RNASEP_CLB RNase P (ribonuclease P) Clostridium 18308982 perfringens RNASEP_EC RNase P (ribonuclease P) Escherichia 16127994 coli RNASEP_RKP RNase P (ribonuclease P) Rickettsia 15603881 prowazekii RNASEP_SA RNase P (ribonuclease P) Staphylococcus 15922990 aureus RNASEP_VBC RNase P (ribonuclease P) Vibrio 15640032 cholerae ICD_CXB icd (isocitrate Coxiella 29732244 dehydrogenase) burnetii IS1111A multi-locus IS1111A Acinetobacter 29732244 insertion element baumannii OMPA_AY485227 ompA (outer membrane protein Rickettsia 40287451 A) prowazekii OMPB_RKP ompB (outer membrane protein Rickettsia 15603881 B) prowazekii GLTA_RKP gltA (citrate synthase) Vibrio 15603881 cholerae TOXR_VBC toxR (transcription Francisella 15640032 regulator toxR) tularensis ASD_FRT asd (Aspartate semialdehyde Francisella 56707187 dehydrogenase) tularensis GALE_FRT galE (UDP-glucose 4- Shigella 56707187 epimerase) flexneri IPAH_SGF ipaH (invasion plasmid Campylobacter 30061571 antigen) jejuni HUPB_CJ hupB (DNA-binding protein Coxiella 15791399 Hu-beta) burnetii AB_MLST Artificial Sequence Artificial Sequenced Concatenation comprising: Sequence* - in- trpE (anthranilate synthase partial gene house component I)) sequences of (SEQ ID adk (adenylate kinase) Acinetobacter NO: mutY (adenine glycosylase) baumannii 1471) fumC (fumarate hydratase) efp (elongation factor p) ppa (pyrophosphate phospho- hydratase MUPR_X75439 mupR (mupriocin resistance Staphylococcus 438226 gene) aureus PARC_X95819 parC (topoisomerase IV) Acinetobacter 1212748 baumannii SED_M28521 sed (enterotoxin D) Staphylococcus 1492109 aureus PLA_AF053945 pla (plasminogen activator) Yersinia 2996216 pestis SEJ_AF053140 sej (enterotoxin J) Staphylococcus 3372540 aureus GYRA_NC000912 gyrA (DNA gyrase subunit A) Mycoplasma 13507739 pneumoniae ACS_NC002516 acsA (Acetyl CoA Synthase) Pseudomonas 15595198 aeruginosa ARO_NC002516 aroE (shikimate 5- Pseudomonas 15595198 dehydrogenase aeruginosa GUA_NC002516 guaA (GMP synthase) Pseudomonas 15595198 aeruginosa MUT_NC002516 mutL (DNA mismatch repair Pseudomonas 15595198 protein) aeruginosa NUO_NC002516 nuoD (NADH dehydrogenase I Pseudomonas 15595198 chain C, D) aeruginosa PPS_NC002516 ppsA (Phosphoenolpyruvate Pseudomonas 15595198 synthase) aeruginosa TRP_NC002516 trpE (Anthranilate Pseudomonas 15595198 synthetase component I) aeruginosa OMP2_NC000117 ompB (outer membrane protein Chlamydia 15604717 B) trachomatis OMPA_NC000117 ompA (outer membrane protein Chlamydia 15604717 B) trachomatis GYRA_NC000117 gyrA (DNA gyrase subunit A) Chlamydia 15604717 trachomatis CTXA_NC002505 ctxA (Cholera toxin A Vibrio 15640032 subunit) cholerae CTXB_NC002505 ctxB (Cholera toxin B Vibrio 15640032 subunit) cholerae FUR_NC002505 fur (ferric uptake regulator Vibrio 15640032 protein) cholerae GAPA_NC_002505 gapA (glyceraldehyde-3- Vibrio 15640032 phosphate dehydrogenase) cholerae GYRB_NC002505 gyrB (DNA gyrase subunit B) Vibrio 15640032 cholerae OMPU_NC002505 ompU (outer membrane Vibrio 15640032 protein) cholerae TCPA_NC002505 tcpA (toxin-coregulated Vibrio 15640032 pilus) cholerae ASPA_NC002163 aspA (aspartate ammonia Campylobacter 15791399 lyase) jejuni GLNA_NC002163 glnA (glutamine synthetase) Campylobacter 15791399 jejuni GLTA_NC002163 gltA (glutamate synthase) Campylobacter 15791399 jejuni GLYA_NC002163 glyA (serine Campylobacter 15791399 hydroxymethyltransferase) jejuni PGM_NC002163 pgm (phosphoglyceromutase) Campylobacter 15791399 jejuni TKT_NC002163 tkt (transketolase) Campylobacter 15791399 jejuni UNCA_NC002163 uncA (ATP synthetase alpha Campylobacter 15791399 chain) jejuni AGR- agr-III (accessory gene Staphylococcus 21281729 III_NC003923 regulator-III) aureus ARCC_NC003923 arcC (carbamate kinase) Staphylococcus 21281729 aureus AROE_NC003923 aroE (shikimate 5- Staphylococcus 21281729 dehydrogenase aureus BSA- bsa-a (glutathione Staphylococcus 21281729 A_NC003923 peroxidase) aureus BSA- bsa-b (epidermin Staphylococcus 21281729 B_NC003923 biosynthesis protein EpiB) aureus GLPF_NC003923 glpF (glycerol transporter) Staphylococcus 21281729 aureus GMK_NC003923 gmk (guanylate kinase) Staphylococcus 21281729 aureus MECI- mecR1 (truncated methicillin Staphylococcus 21281729 R_NC003923 resistance protein) aureus PTA_NC003923 pta (phosphate Staphylococcus 21281729 acetyltransferase) aureus PVLUK_NC003923 pvluk (Panton-Valentine Staphylococcus 21281729 leukocidin chain F aureus precursor) SA442_NC003923 sa442 gene Staphylococcus 21281729 aureus SEA_NC003923 sea (staphylococcal Staphylococcus 21281729 enterotoxin A precursor) aureus SEC_NC003923 sec4 (enterotoxin type C Staphylococcus 21281729 precursor) aureus TPI_NC003923 tpi (triosephosphate Staphylococcus 21281729 isomerase) aureus YQI_NC003923 yqi (acetyl-CoA C- Staphylococcus 21281729 acetyltransferase homologue) aureus GALE_AF513299 galE (galactose epimerase) Francisella 23506418 tularensis VVHA_NC004460 vVhA (cytotoxin, cytolysin Vibrio 27366463 precursor) vulnificus TDH_NC004605 tdh (thermostable direct Vibrio 28899855 hemolysin A) parahaemolyticus AGR- agr-II (accessory gene Staphylococcus 29165615 II_NC002745 regulator-II) aureus PARC_NC003997 parC (topoisomerase IV) Bacillus 30260195 anthracis GYRA_AY291534 gyrA (DNA gyrase subunit A) Bacillus 31323274 anthracis AGR- agr-I (accessory gene Staphylococcus 46019543 I_AJ617706 regulator-I) aureus AGR- agr-IV (accessory gene Staphylococcus 46019563 IV_AJ617711 regulator-III) aureus BLAZ_NC002952 blaZ (beta lactamase III) Staphylococcus 49482253 aureus ERMA_NC002952 ermA (rRNA methyltransferase Staphylococcus 49482253 A) aureus ERMB_Y13600 ermB (rRNA methyltransferase Staphylococcus 49482253 B) aureus SEA- sea (staphylococcal Staphylococcus 49482253 SEE_NC002952 enterotoxin A precursor) aureus SEA- sea (staphylococcal Staphylococcus 49482253 SEE_NC002952 enterotoxin A precursor) aureus SEE_NC002952 sea (staphylococcal Staphylococcus 49482253 enterotoxin A precursor) aureus SEH_NC002953 seh (staphylococcal Staphylococcus 49484912 enterotoxin H) aureus ERMC_NC005908 ermC (rRNA methyltransferase Staphylococcus 49489772 C) aureus MUTS_AY698802 mutS (DNA mismatch repair Shigella 52698233 protein) boydii NUC_NC002758 nuc (staphylococcal Staphylococcus 57634611 nuclease) aureus SEB_NC002758 seb (enterotoxin type B Staphylococcus 57634611 precursor) aureus SEG_NC002758 seg (staphylococcal Staphylococcus 57634611 enterotoxin G) aureus SEI_NC002758 sei (staphylococcal Staphylococcus 57634611 enterotoxin I) aureus TSST_NC002758 tsst (toxic shock syndrome Staphylococcus 57634611 toxin-1) aureus TUFB_NC002758 tufB (Elongation factor Tu) Staphylococcus 57634611 aureus Note: artificial reference sequences represent concatenations of partial gene extractions from the indicated reference gi number. Partial sequences were used to create the concatenated sequence because complete gene sequences were not necessary for primer design.

Example 2 Sample Preparation and PCR

Genomic DNA was prepared from samples using the DNeasy Tissue Kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocols.

All PCR reactions were assembled in 50 μL reaction volumes in a 96-well microtiter plate format using a Packard MPII liquid handling robotic platform and M.J. Dyad thermocyclers (MJ research, Waltham, Mass.) or Eppendorf Mastercycler thermocyclers (Eppendorf, Westbury, N.Y.). The PCR reaction mixture consisted of 4 units of Amplitaq Gold, 1× buffer II (Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl₂, 0.4 M betaine, 800 μM dNTP mixture and 250 nM of each primer. The following typical PCR conditions were used: 95° C. for 10 min followed by 8 cycles of 95° C. for 30 seconds, 48° C. for 30 seconds, and 72° C. 30 seconds with the 48° C. annealing temperature increasing 0.9° C. with each of the eight cycles. The PCR was then continued for 37 additional cycles of 95° C. for 15 seconds, 56° C. for 20 seconds, and 72° C. 20 seconds.

Example 3 Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads

For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, 25 μl of a 2.5 mg/mL suspension of BioClone amine terminated superparamagnetic beads were added to 25 to 50 μl of a PCR (or RT-PCR) reaction containing approximately 10 pM of a typical PCR amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed three times with 50 mM ammonium bicarbonate/50% MeOH or 100 mM ammonium bicarbonate/50% MeOH, followed by three more washes with 50% MeOH. The bound PCR amplicon was eluted with a solution of 25 mM piperidine, 25 mM imidazole, 35% MeOH which included peptide calibration standards.

Example 4 Mass Spectrometry and Base Composition Analysis

The ESI-FTICR mass spectrometer is based on a Bruker Daltonics (Billerica, Mass.) Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer that employs an actively shielded 7 Tesla superconducting magnet. The active shielding constrains the majority of the fringing magnetic field from the superconducting magnet to a relatively small volume. Thus, components that might be adversely affected by stray magnetic fields, such as CRT monitors, robotic components, and other electronics, can operate in close proximity to the FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT 4.0 operating system. Sample aliquots, typically 15 μl, were extracted directly from 96-well microtiter plates using a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, N.C.) triggered by the FTICR data station. Samples were injected directly into a 10 μl sample loop integrated with a fluidics handling system that supplies the 100 μl/hr flow rate to the ESI source. Ions were formed via electrospray ionization in a modified Analytica (Branford, Conn.) source employing an off axis, grounded electrospray probe positioned approximately 1.5 cm from the metallized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary was biased at 6000 V relative to the ESI needle during data acquisition. A counter-current flow of dry N₂ was employed to assist in the desolvation process. Ions were accumulated in an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode, prior to injection into the trapped ion cell where they were mass analyzed. Ionization duty cycles greater than 99% were achieved by simultaneously accumulating ions in the external ion reservoir during ion detection. Each detection event consisted of 1 M data points digitized over 2.3 s. To improve the signal-to-noise ratio (S/N), 32 scans were co-added for a total data acquisition time of 74 s.

The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOF™. Ions from the ESI source undergo orthogonal ion extraction and are focused in a reflectron prior to detection. The TOF and FTICR are equipped with the same automated sample handling and fluidics described above. Ions are formed in the standard MicroTOF™ ESI source that is equipped with the same off-axis sprayer and glass capillary as the FTICR ESI source. Consequently, source conditions were the same as those described above. External ion accumulation was also employed to improve ionization duty cycle during data acquisition. Each detection event on the TOF was comprised of 75,000 data points digitized over 75 μs.

The sample delivery scheme allows sample aliquots to be rapidly injected into the electrospray source at high flow rate and subsequently be electrosprayed at a much lower flow rate for improved ESI sensitivity. Prior to injecting a sample, a bolus of buffer was injected at a high flow rate to rinse the transfer line and spray needle to avoid sample contamination/carryover. Following the rinse step, the autosampler injected the next sample and the flow rate was switched to low flow. Following a brief equilibration delay, data acquisition commenced. As spectra were co-added, the autosampler continued rinsing the syringe and picking up buffer to rinse the injector and sample transfer line. In general, two syringe rinses and one injector rinse were required to minimize sample carryover. During a routine screening protocol a new sample mixture was injected every 106 seconds. More recently a fast wash station for the syringe needle has been implemented which, when combined with shorter acquisition times, facilitates the acquisition of mass spectra at a rate of just under one spectrum/minute.

Raw mass spectra were post-calibrated with an internal mass standard and deconvoluted to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides. Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 500 molecules per well. Calibration methods are commonly owned and disclosed in PCT Publication Number WO 2005/098047 which is incorporated herein by reference in entirety.

Example 5 De Novo Determination of Base Composition of Amplification Products Using Molecular Mass Modified Deoxynucleotide Triphosphates

Because the molecular masses of the four natural nucleobases have a relatively narrow molecular mass range (A=313.058, G=329.052, C=289.046, T=304.046—See Table 4), a persistent source of ambiguity in assignment of base composition can occur as follows: two nucleic acid strands having different base composition may have a difference of about 1 Da when the base composition difference between the two strands is G

A (−15.994) combined with C

T (+15.000). For example, one 99-mer nucleic acid strand having a base composition of A₂₇G₃₀C₂₁T₂₁ has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A₂₆G₃₁C₂₂T₂₀ has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor.

The methods provide for a means for removing this theoretical 1 Da uncertainty factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural nucleobases. The term “nucleobase” as used herein is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).

Addition of significant mass to one of the 4 nucleobases (dNTPs) in an amplification reaction, or in the primers themselves, will result in a significant difference in mass of the resulting amplification product (significantly greater than 1 Da) arising from ambiguities arising from the G

A combined with C

T event (Table 4). Thus, the same the G

A (−15.994) event combined with 5-Iodo-C

T (−110.900) event would result in a molecular mass difference of 126.894. If the molecular mass of the base composition A₂₇G₃₀ 5-Iodo-C₂₁T₂₁ (33422.958) is compared with A₂₆G₃₁ 5-Iodo-C₂₂T₂₀, (33549.852) the theoretical molecular mass difference is +126.894. The experimental error of a molecular mass measurement is not significant with regard to this molecular mass difference. Furthermore, the only base composition consistent with a measured molecular mass of the 99-mer nucleic acid is A₂₇G₃₀ 5-Iodo-C₂₁T₂₁. In contrast, the analogous amplification without the mass tag has 18 possible base compositions.

TABLE 4 Molecular Masses of Natural Nucleobases and the Mass-Modified Nucleobase 5-Iodo-C and Molecular Mass Differences Resulting from Transitions Molecular Δ Molecular Nucleobase Mass Transition Mass A 313.058 A-->T −9.012 A 313.058 A-->C −24.012 A 313.058 A-->5- 101.888 Iodo-C A 313.058 A-->G 15.994 T 304.046 T-->A 9.012 T 304.046 T-->C −15.000 T 304.046 T-->5- 110.900 Iodo-C T 304.046 T-->G 25.006 C 289.046 C-->A 24.012 C 289.046 C-->T 15.000 C 289.046 C-->G 40.006 5-Iodo-C 414.946 5-Iodo-C-->A −101.888 5-Iodo-C 414.946 5-Iodo-C-->T −110.900 5-Iodo-C 414.946 5-Iodo-C-->G −85.894 G 329.052 G-->A −15.994 G 329.052 G-->T −25.006 G 329.052 G-->C −40.006 G 329.052 G-->5- 85.894 Iodo-C

Mass spectra of bioagent-identifying amplicons were analyzed independently using a maximum-likelihood processor, such as is widely used in radar signal processing. This processor, referred to as GenX, first makes maximum likelihood estimates of the input to the mass spectrometer for each primer by running matched filters for each base composition aggregate on the input data. This includes the GenX response to a calibrant for each primer.

The algorithm emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database is used to define the mass base count matched filters. The database contains the sequences of known bacterial bioagents and includes threat organisms as well as benign background organisms. The latter is used to estimate and subtract the spectral signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. The maximum likelihood process is applied to this “cleaned up” data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.

The amplitudes of all base compositions of bioagent-identifying amplicons for each primer are calibrated and a final maximum likelihood amplitude estimate per organism is made based upon the multiple single primer estimates. Models of all system noise are factored into this two-stage maximum likelihood calculation. The processor reports the number of molecules of each base composition contained in the spectra. The quantity of amplification product corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction.

Base count blurring can be carried out as follows. “Electronic PCR” can be conducted on nucleotide sequences of the desired bioagents to obtain the different expected base counts that could be obtained for each primer pair. See for example, ncbi.nlm.nih.gov/sutils/e-pcr/; Schuler, Genome Res. 7:541-50, 1997. In one illustrative embodiment, one or more spreadsheets, such as Microsoft Excel workbooks contain a plurality of worksheets. First in this example, there is a worksheet with a name similar to the workbook name; this worksheet contains the raw electronic PCR data. Second, there is a worksheet named “filtered bioagents base count” that contains bioagent name and base count; there is a separate record for each strain after removing sequences that are not identified with a genus and species and removing all sequences for bioagents with less than 10 strains. Third, there is a worksheet, “Sheet1” that contains the frequency of substitutions, insertions, or deletions for this primer pair. This data is generated by first creating a pivot table from the data in the “filtered bioagents base count” worksheet and then executing an Excel VBA macro. The macro creates a table of differences in base counts for bioagents of the same species, but different strains. One of ordinary skill in the art may understand additional pathways for obtaining similar table differences without undo experimentation.

Application of an exemplary script, involves the user defining a threshold that specifies the fraction of the strains that are represented by the reference set of base counts for each bioagent. The reference set of base counts for each bioagent may contain as many different base counts as are needed to meet or exceed the threshold. The set of reference base counts is defined by taking the most abundant strain's base type composition and adding it to the reference set and then the next most abundant strain's base type composition is added until the threshold is met or exceeded. The current set of data was obtained using a threshold of 55%, which was obtained empirically.

For each base count not included in the reference base count set for that bioagent, the script then proceeds to determine the manner in which the current base count differs from each of the base counts in the reference set. This difference may be represented as a combination of substitutions, Si=Xi, and insertions, Ii=Yi, or deletions, Di=Zi. If there is more than one reference base count, then the reported difference is chosen using rules that aim to minimize the number of changes and, in instances with the same number of changes, minimize the number of insertions or deletions. Therefore, the primary rule is to identify the difference with the minimum sum (Xi+Yi) or (Xi+Zi), e.g., one insertion rather than two substitutions. If there are two or more differences with the minimum sum, then the one that will be reported is the one that contains the most substitutions.

Differences between a base count and a reference composition are categorized as one, two, or more substitutions, one, two, or more insertions, one, two, or more deletions, and combinations of substitutions and insertions or deletions. The different classes of nucleobase changes and their probabilities of occurrence have been delineated in U.S. Patent Application Publication No. 2004209260 (U.S. application Ser. No. 10/418,514) which is incorporated herein by reference in entirety.

Example 6 Use of Broad Range Survey and Division Wide Primer Pairs for Identification of Bacteria in an Epidemic Surveillance Investigation

This investigation employed a set of 16 primer pairs which is herein designated the “surveillance primer set” and comprises broad range survey primer pairs, division wide primer pairs and a single Bacillus clade primer pair. The surveillance primer set is shown in Table 5 and consists of primer pairs originally listed in Table 2. This surveillance set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Primer pair 449 (non-T modified) has been modified twice. Its predecessors are primer pairs 70 and 357, displayed below in the same row. Primer pair 360 has also been modified twice and its predecessors are primer pairs 17 and 118.

TABLE 5 Bacterial Primer Pairs of the Surveillance Primer Set Forward Reverse Primer Primer Primer (SEQ (SEQ Pair ID ID Target No. Forward Primer Name NO:) Reverse Primer Name NO:) Gene 346 16S_EC_713_732_TMOD_F 202 16S_EC_789_809_TMOD_R 1110 16S rRNA 10 16S_EC_713_732_F 21 16S_EC_789_809 798 16S rRNA 347 16S_EC_785_806_TMOD_F 560 16S_EC_880_897_TMOD_R 1278 16S rRNA 11 16S_EC_785_806_F 118 16S_EC_880_897_R 830 16S rRNA 348 16S_EC_960_981_TMOD_F 706 16S_EC_1054_1073_TMOD_R 895 16S rRNA 14 16S_EC_960_981_F 672 16S_EC_1054_1073_R 735 16S rRNA 349 23S_EC_1826_1843_TMOD_F 401 23S_EC_1906_1924_TMOD_R 1156 23S rRNA 16 23S_EC_1826_1843_F 80 23S_EC_1906_1924_R 805 23S rRNA 352 INFB_EC_1365_1393_TMOD_F 687 INFB_EC_1439_1467_TMOD_R 1411 infB 34 INFB_EC_1365_1393_F 524 INFB_EC_1439_1467_R 1248 infB 354 RPOC_EC_2218_2241_TMOD_F 405 RPOC_EC_2313_2337_TMOD_R 1072 rpoC 52 RPOC_EC_2218_2241_F 81 RPOC_EC_2313_2337_R 790 rpoC 355 SSPE_BA_115_137_TMOD_F 255 SSPE_BA_197_222_TMOD_R 1402 sspE 58 SSPE_BA_115_137_F 45 SSPE_BA_197_222_R 1201 sspE 356 RPLB_EC_650_679_TMOD_F 232 RPLB_EC_739_762_TMOD_R 592 rplB 66 RPLB_EC_650_679_F 98 RPLB_EC_739_762_R 999 rplB 358 VALS_EC_1105_1124_TMOD_F 385 VALS_EC_1195_1218_TMOD_R 1093 valS 71 VALS_EC_1105_1124_F 77 VALS_EC_1195_1218_R 795 valS 359 RPOB_EC_1845_1866_TMOD_F 659 RPOB_EC_1909_1929_TMOD_R 1250 rpoB 72 RPOB_EC_1845_1866_F 233 RPOB_EC_1909_1929_R 825 rpoB 360 23S_EC_2646_2667_TMOD_F 409 23S_EC_2745_2765_TMOD_R 1434 23S rRNA 118 23S_EC_2646_2667_F 84 23S_EC_2745_2765_R 1389 23S rRNA 17 23S_EC_2645_2669_F 408 23S_EC_2744_2761_R 1252 23S rRNA 361 16S_EC_1090_1111_2_TMOD_F 697 16S_EC_1175_1196_TMOD_R 1398 16S rRNA 3 16S_EC_1090_1111_2_F 651 16S_EC_1175_1196_R 1159 16S rRNA 362 RPOB_EC_3799_3821_TMOD_F 581 RPOB_EC_3862_3888_TMOD_R 1325 rpoB 289 RPOB_EC_3799_3821_F 124 RPOB_EC_3862_3888_R 840 rpoB 363 RPOC_EC_2146_2174_TMOD_F 284 RPOC_EC_2227_2245_TMOD_R 898 rpoC 290 RPOC_EC_2146_2174_F 52 RPOC_EC_2227_2245_R 736 rpoC 367 TUFB_EC_957_979_TMOD_F 308 TUFB_EC_1034_1058_TMOD_R 1276 tufB 293 TUFB_EC_957_979_F 55 TUFB_EC_1034_1058_R 829 tufB 449 RPLB_EC_690_710_F 309 RPLB_EC_737_758_R 1336 rplB 357 RPLB_EC_688_710_TMOD_F 296 RPLB_EC_736_757_TMOD_R 1337 rplB 67 RPLB_EC_688_710_F 54 RPLB_EC_736_757_R 842 rplB

The 16 primer pairs of the surveillance set are used to produce bioagent identifying amplicons whose base compositions are sufficiently different amongst all known bacteria at the species level to identify, at a reasonable confidence level, any given bacterium at the species level. As shown in Tables 6A-E, common respiratory bacterial pathogens can be distinguished by the base compositions of bioagent identifying amplicons obtained using the 16 primer pairs of the surveillance set. In some cases, triangulation identification improves the confidence level for species assignment. For example, nucleic acid from Streptococcus pyogenes can be amplified by nine of the sixteen surveillance primer pairs and Streptococcus pneumoniae can be amplified by ten of the sixteen surveillance primer pairs. The base compositions of the bioagent identifying amplicons are identical for only one of the analogous bioagent identifying amplicons and differ in all of the remaining analogous bioagent identifying amplicons by up to four bases per bioagent identifying amplicon. The resolving power of the surveillance set was confirmed by determination of base compositions for 120 isolates of respiratory pathogens representing 70 different bacterial species and the results indicated that natural variations (usually only one or two base substitutions per bioagent identifying amplicon) amongst multiple isolates of the same species did not prevent correct identification of major pathogenic organisms at the species level.

Bacillus anthracis is a well known biological warfare agent which has emerged in domestic terrorism in recent years. Since it was envisioned to produce bioagent identifying amplicons for identification of Bacillus anthracis, additional drill-down analysis primers were designed to target genes present on virulence plasmids of Bacillus anthracis so that additional confidence could be reached in positive identification of this pathogenic organism. Three drill-down analysis primers were designed and are listed in Tables 2 and 6. In Table 6, the drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

TABLE 6 Drill-Down Primer Pairs for Confirmation of Identification of Bacillus anthracis Forward Primer Reverse Primer (SEQ Primer Pair ID (SEQ ID Target No. Forward Primer Name NO:) Reverse Primer Name NO:) Gene 350 CAPC_BA_274_303_TMOD_F 476 CAPC_BA_349_376_TMOD_R 1314 capC 24 CAPC_BA_274_303_F 109 CAPC_BA_349_376_R 837 capC 351 CYA_BA_1353_1379_TMOD_F 355 CYA_BA_1448_1467_TMOD_R 1423 cyA 30 CYA_BA_1353_1379_F 64 CYA_BA_1448_1467_R 1342 cyA 353 LEF_BA_756_781_TMOD_F 220 LEF_BA_843_872_TMOD_R 1394 lef 37 LEF_BA_756_781_F 26 LEF_BA_843_872_R 1135 lef

Phylogenetic coverage of bacterial space of the sixteen surveillance primers of Table 5 and the three Bacillus anthracis drill-down primers of Table 6 is shown in FIG. 3 which lists common pathogenic bacteria. FIG. 3 is not meant to be comprehensive in illustrating all species identified by the primers. Only pathogenic bacteria are listed as representative examples of the bacterial species that can be identified by the primers and methods disclosed herein. Nucleic acid of groups of bacteria enclosed within the polygons of FIG. 3 can be amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand corner of each polygon. Primer coverage for polygons within polygons is additive. As an illustrative example, bioagent identifying amplicons can be obtained for Chlamydia trachomatis by amplification with, for example, primer pairs 346-349, 360 and 361, but not with any of the remaining primers of the surveillance primer set. On the other hand, bioagent identifying amplicons can be obtained from nucleic acid originating from Bacillus anthracis (located within 5 successive polygons) using, for example, any of the following primer pairs: 346-349, 360, 361 (base polygon), 356, 449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350, 351 and 353 (fifth polygon). Multiple coverage of a given organism with multiple primers provides for increased confidence level in identification of the organism as a result of enabling broad triangulation identification.

In Tables 7A-E, base compositions of respiratory pathogens for primer target regions are shown. Two entries in a cell, represent variation in ribosomal DNA operons. The most predominant base composition is shown first and the minor (frequently a single operon) is indicated by an asterisk (*). Entries with NO DATA mean that the primer would not be expected to prime this species due to mismatches between the primer and target region, as determined by theoretical PCR.

TABLE 7A Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 346, 347 and 348 Primer Primer Primer 346 347 348 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella MGH78578 [29 32 25 [23 38 28 [26 32 28 pneumoniae 13] 26] 30] [29 31 25 [23 37 28 [26 31 28 13]* 26]* 30]* Yersinia CO-92 Biovar [29 32 25 [22 39 28 [29 30 28 pestis Orientalis 13] 26] 29] [30 30 27 29]* Yersinia KIM5 P12 [29 32 25 [22 39 28 [29 30 28 pestis (Biovar 13] 26] 29] Mediaevalis) Yersinia 91001 [29 32 25 [22 39 28 [29 30 28 pestis 13] 26] 29] [30 30 27 29]* Haemophilus KW20 [28 31 23 [24 37 25 [29 30 28 influenzae 17] 27] 29] Pseudomonas PAO1 [30 31 23 [26 36 29 [26 32 29 aeruginosa 15] 24] [27 29] 36 29 23]* Pseudomonas Pf0-1 [30 31 23 [26 35 29 [28 31 28 fluorescens 15] 25] 29] Pseudomonas KT2440 [30 31 23 [28 33 27 [27 32 29 putida 15] 27] 28] Legionella Philadelphia-1 [30 30 24 [33 33 23 [29 28 28 pneumophila 15] 27] 31] Francisella schu 4 [32 29 22 [28 38 26 [25 32 28 tularensis 16] 26] 31] Bordetella Tohama I [30 29 24 [23 37 30 [30 32 30 pertussis 16] 24] 26] Burkholderia J2315 [29 29 27 [27 32 26 [27 36 31 cepacia 14] 29] 24] [20 42 35 19]* Burkholderia K96243 [29 29 27 [27 32 26 [27 36 31 pseudomallei 14] 29] 24] Neisseria FA 1090, [29 28 24 [27 34 26 [24 36 29 gonorrhoeae ATCC 18] 28] 27] 700825 Neisseria MC58 [29 28 26 [27 34 27 [25 35 30 meningitidis (serogroup B) 16] 27] 26] Neisseria serogroup C, [29 28 26 [27 34 27 [25 35 30 meningitidis FAM18 16] 27] 26] Neisseria Z2491 [29 28 26 [27 34 27 [25 35 30 meningitidis (serogroup A) 16] 27] 26] Chlamydophila TW-183 [31 27 22 NO DATA [32 27 27 pneumoniae 19] 29] Chlamydophila AR39 [31 27 22 NO DATA [32 27 27 pneumoniae 19] 29] Chlamydophila CWL029 [31 27 22 NO DATA [32 27 27 pneumoniae 19] 29] Chlamydophila J138 [31 27 22 NO DATA [32 27 27 pneumoniae 19] 29] Corynebacterium NCTC13129 [29 34 21 [22 38 31 [22 33 25 diphtheriae 15] 25] 34] Mycobacterium k10 [27 36 21 [22 37 30 [21 36 27 avium 15] 28] 30] Mycobacterium 104 [27 36 21 [22 37 30 [21 36 27 avium 15] 28] 30] Mycobacterium CSU#93 [27 36 21 [22 37 30 [21 36 27 tuberculosis 15] 28] 30] Mycobacterium CDC 1551 [27 36 21 [22 37 30 [21 36 27 tuberculosis 15] 28] 30] Mycobacterium H37Rv (lab [27 36 21 [22 37 30 [21 36 27 tuberculosis strain) 15] 28] 30] Mycoplasma M129 [31 29 19 NO DATA NO pneumoniae 20] DATA Staphylococcus MRSA252 [27 30 21 [25 35 30 [30 29 30 aureus 21] 26] 29] [29 31 30 29]* Staphylococcus MSSA476 [27 30 21 [25 35 30 [30 29 30 aureus 21] 26] 29] [30 29 29 30]* Staphylococcus COL [27 30 21 [25 35 30 [30 29 30 aureus 21] 26] 29] [30 29 29 30]* Staphylococcus Mu50 [27 30 21 [25 35 30 [30 29 30 aureus 21] 26] 29] [30 29 29 30]* Staphylococcus MW2 [27 30 21 [25 35 30 [30 29 30 aureus 21] 26] 29] [30 29 29 30]* Staphylococcus N315 [27 30 21 [25 35 30 [30 29 30 aureus 21] 26] 29] [30 29 29 30]* Staphylococcus NCTC 8325 [27 30 21 [25 35 30 [30 29 30 aureus 21] 26] [25 29] [30 35 31 26]* 29 29 30] Streptococcus NEM316 [26 32 23 [24 36 31 [25 32 29 agalactiae 18] 25] [24 30] 36 30 26]* Streptococcus NC_002955 [26 32 23 [23 37 31 [29 30 25 equi 18] 25] 32] Streptococcus MGAS8232 [26 32 23 [24 37 30 [25 31 29 pyogenes 18] 25] 31] Streptococcus MGAS315 [26 32 23 [24 37 30 [25 31 29 pyogenes 18] 25] 31] Streptococcus SSI-1 [26 32 23 [24 37 30 [25 31 29 pyogenes 18] 25] 31] Streptococcus MGAS10394 [26 32 23 [24 37 30 [25 31 29 pyogenes 18] 25] 31] Streptococcus Manfredo (M5) [26 32 23 [24 37 30 [25 31 29 pyogenes 18] 25] 31] Streptococcus SF370 (M1) [26 32 23 [24 37 30 [25 31 29 pyogenes 18] 25] 31] Streptococcus 670 [26 32 23 [25 35 28 [25 32 29 pneumoniae 18] 28] 30] Streptococcus R6 [26 32 23 [25 35 28 [25 32 29 pneumoniae 18] 28] 30] Streptococcus TIGR4 [26 32 23 [25 35 28 [25 32 30 pneumoniae 18] 28] 29] Streptococcus NCTC7868 [25 33 23 [24 36 31 [25 31 29 gordonii 18] 25] 31] Streptococcus NCTC 12261 [26 32 23 [25 35 30 [25 32 29 mitis 18] 26] 30] [24 31 35 29]* Streptococcus UA159 [24 32 24 [25 37 30 [28 31 26 mutans 19] 24] 31]

TABLE 7B Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 349, 360, and 356 Primer 349 Primer 360 Primer 356 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella MGH78578 [25 31 25 [33 37 25 NO DATA pneumoniae 22] 27] Yersinia CO-92 Biovar [25 31 27 [34 35 25 NO DATA pestis Orientalis 20] [25 28] 32 26 20]* Yersinia KIM5 P12 [25 31 2720] [34 35 25 NO DATA pestis (Biovar Mediaevalis) [25 32 26 20]* 28] Yersinia 91001 [25 31 27 [34 35 25 NO DATA pestis 20] 28] Haemophilus KW20 [28 28 25 [32 38 25 NO DATA influenzae 20] 27] Pseudomonas PAO1 [24 31 26 [31 36 27 NO DATA aeruginosa 20] 27] [31 36 27 28]* Pseudomonas Pf0-1 NO DATA [30 37 27 28] NO DATA fluorescens [30 37 27 28] Pseudomonas KT2440 [24 31 26 [30 37 27 NO DATA putida 20] 28] Legionella Philadelphia-1 [23 30 25 [30 39 29 NO DATA pneumophila 23] 24] Francisella schu 4 [26 31 25 [32 36 27 NO DATA tularensis 19] 27] Bordetella Tohama I [21 29 24 [33 36 26 NO DATA pertussis 18] 27] Burkholderia J2315 [23 27 22 [31 37 28 NO DATA cepacia 20] 26] Burkholderia K96243 [23 27 22 [31 37 28 NO DATA pseudomallei 20] 26] Neisseria FA 1090, ATCC 700825 [24 27 24 [34 37 25 NO DATA gonorrhoeae 17] 26] Neisseria MC58 (serogroup B) [25 27 22 [34 37 25 NO DATA meningitidis 18] 26] Neisseria serogroup C, [25 26 23 [34 37 25 NO DATA meningitidis FAM18 18] 26] Neisseria Z2491 [25 26 23 [34 37 25 NO DATA meningitidis (serogroup A) 18] 26] Chlamydophila TW-183 [30 28 27 NO DATA NO DATA pneumoniae 18] Chlamydophila AR39 [30 28 27 NO DATA NO DATA pneumoniae 18] Chlamydophila CWL029 [30 28 27 NO DATA NO DATA pneumoniae 18] Chlamydophila J138 [30 28 27 NO DATA NO DATA pneumoniae 18] Corynebacterium NCTC13129 NO DATA [29 40 28 NO DATA diphtheriae 25] Mycobacterium k10 NO DATA [33 35 32 NO DATA avium 22] Mycobacterium 104 NO DATA [33 35 32 NO DATA avium 22] Mycobacterium CSU#93 NO DATA [30 36 34 NO DATA tuberculosis 22] Mycobacterium CDC 1551 NO DATA [30 36 34 NO DATA tuberculosis 22] Mycobacterium H37Rv (lab NO DATA [30 36 34 NO DATA tuberculosis strain) 22] Mycoplasma M129 [28 30 24 [34 31 29 NO DATA pneumoniae 19] 28] Staphylococcus MRSA252 [26 30 25 [31 38 24 [33 30 31 aureus 20] 29] 27] Staphylococcus MSSA476 [26 30 25 [31 38 24 [33 30 31 aureus 20] 29] 27] Staphylococcus COL [26 30 25 [31 38 24 [33 30 31 aureus 20] 29] 27] Staphylococcus Mu50 [26 30 25 [31 38 24 [33 30 31 aureus 20] 29] 27] Staphylococcus MW2 [26 30 25 [31 38 24 [33 30 31 aureus 20] 29] 27] Staphylococcus N315 [26 30 25 [31 38 24 [33 30 31 aureus 20] 29] 27] Staphylococcus NCTC 8325 [26 30 25 [31 38 24 [33 30 31 aureus 20] 29] 27] Streptococcus NEM316 [28 31 22 [33 37 24 [37 30 28 agalactiae 20] 28] 26] Streptococcus NC_002955 [28 31 23 [33 38 24 [37 31 28 equi 19] 27] 25] Streptococcus MGAS8232 [28 31 23 [33 37 24 [38 31 29 pyogenes 19] 28] 23] Streptococcus MGAS315 [28 31 23 [33 37 24 [38 31 29 pyogenes 19] 28] 23] Streptococcus SSI-1 [28 31 23 [33 37 24 [38 31 29 pyogenes 19] 28] 23] Streptococcus MGAS10394 [28 31 23 [33 37 24 [38 31 29 pyogenes 19] 28] 23] Streptococcus Manfredo (M5) [28 31 23 [33 37 24 [38 31 29 pyogenes 19] 28] 23] Streptococcus SF370 (M1) [28 31 23 [33 37 24 [38 31 29 pyogenes 19] [28 28] 23] 31 22 20]* Streptococcus 670 [28 31 22 [34 36 24 [37 30 29 pneumoniae 20] 28] 25] Streptococcus R6 [28 31 22 [34 36 24 [37 30 29 pneumoniae 20] 28] 25] Streptococcus TIGR4 [28 31 22 [34 36 24 [37 30 29 pneumoniae 20] 28] 25] Streptococcus NCTC7868 [28 32 23 [34 36 24 [36 31 29 gordonii 20] 28] 25] Streptococcus NCTC 12261 [28 31 22 [34 36 24 [37 30 29 mitis 20] [29 28] 25] 30 22 20]* Streptococcus UA159 [26 32 23 [34 37 24 NO DATA mutans 22] 27]

TABLE 7C Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 449, 354, and 352 Primer 449 Primer 354 Primer 352 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella MGH78578 NO DATA [27 33 36 NO DATA pneumoniae 26] Yersinia CO-92 Biovar NO DATA [29 31 33 [32 28 20 pestis Orientalis 29] 25] Yersinia KIM5 P12 NO DATA [29 31 33 [32 28 20 pestis (Biovar Mediaevalis) 29] 25] Yersinia pestis 91001 NO DATA [29 31 33 29] NO DATA Haemophilus KW20 NO DATA [30 29 31 NO DATA influenzae 32] Pseudomonas PAO1 NO DATA [26 33 39 NO DATA aeruginosa 24] Pseudomonas Pf0-1 NO DATA [26 33 34 NO DATA fluorescens 29] Pseudomonas KT2440 NO DATA [25 34 36 NO DATA putida 27] Legionella Philadelphia-1 NO DATA NO DATA NO DATA pneumophila Francisella schu 4 NO DATA [33 32 25 NO DATA tularensis 32] Bordetella Tohama I NO DATA [26 33 39 NO DATA pertussis 24] Burkholderia J2315 NO DATA [25 37 33 NO DATA cepacia 27] Burkholderia K96243 NO DATA [25 37 34 NO DATA pseudomallei 26] Neisseria FA 1090, ATCC [17 23 22 [29 31 32 NO DATA gonorrhoeae 700825 10] 30] Neisseria MC58 (serogroup NO DATA [29 30 32 NO DATA meningitidis B) 31] Neisseria serogroup C, NO DATA [29 30 32 NO DATA meningitidis FAM18 31] Neisseria Z2491 NO DATA [29 30 32 NO DATA meningitidis (serogroup A) 31] Chlamydophila TW-183 NO DATA NO DATA NO DATA pneumoniae Chlamydophila AR39 NO DATA NO DATA NO DATA pneumoniae Chlamydophila CWL029 NO DATA NO DATA NO DATA pneumoniae Chlamydophila J138 NO DATA NO DATA NO DATA pneumoniae Corynebacterium NCTC13129 NO DATA NO DATA NO DATA diphtheriae Mycobacterium avium k10 NO DATA NO DATA NO DATA Mycobacterium avium 104 NO DATA NO DATA NO DATA Mycobacterium CSU#93 NO DATA NO DATA NO DATA tuberculosis Mycobacterium CDC 1551 NO DATA NO DATA NO DATA tuberculosis Mycobacterium H37Rv (lab NO DATA NO DATA NO DATA tuberculosis strain) Mycoplasma M129 NO DATA NO DATA NO DATA pneumoniae Staphylococcus MRSA252 [17 20 21 [30 27 30 [36 24 19 aureus 17] 35] 26] Staphylococcus MSSA476 [17 20 21 [30 27 30 [36 24 19 aureus 17] 35] 26] Staphylococcus COL [17 20 21 [30 27 30 [35 24 19 aureus 17] 35] 27] Staphylococcus Mu50 [17 20 21 [30 27 30 [36 24 19 aureus 17] 35] 26] Staphylococcus MW2 [17 20 21 [30 27 30 [36 24 19 aureus 17] 35] 26] Staphylococcus N315 [17 20 21 [30 27 30 [36 24 19 aureus 17] 35] 26] Staphylococcus NCTC 8325 [17 20 21 [30 27 30 [35 24 19 aureus 17] 35] 27] Streptococcus NEM316 [22 20 19 [26 31 27 [29 26 22 agalactiae 14] 38] 28] Streptococcus equi NC_002955 [22 21 19 13] NO DATA NO DATA Streptococcus MGAS8232 [23 21 19 [24 32 30 NO DATA pyogenes 12] 36] Streptococcus MGAS315 [23 21 19 [24 32 30 NO DATA pyogenes 12] 36] Streptococcus SSI-1 [23 21 19 [24 32 30 NO DATA pyogenes 12] 36] Streptococcus MGAS10394 [23 21 19 [24 32 30 NO DATA pyogenes 12] 36] Streptococcus Manfredo (M5) [23 21 19 [24 32 30 NO DATA pyogenes 12] 36] Streptococcus SF370 (M1) [23 21 19 [24 32 30 NO DATA pyogenes 12] 36] Streptococcus 670 [22 20 19 [25 33 29 [30 29 21 pneumoniae 14] 35] 25] Streptococcus R6 [22 20 19 [25 33 29 [30 29 21 pneumoniae 14] 35] 25] Streptococcus TIGR4 [22 20 19 [25 33 29 [30 29 21 pneumoniae 14] 35] 25] Streptococcus NCTC7868 [21 21 19 NO DATA [29 26 22 gordonii 14] 28] Streptococcus NCTC 12261 [22 20 19 [26 30 32 NO DATA mitis 14] 34] Streptococcus mutans UA159 NO DATA NO DATA NO DATA

TABLE 7D Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 355, 358, and 359 Primer 355 Primer 358 Primer 359 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella MGH78578 NO DATA [24 39 33 20] [25 21 24 17] pneumoniae Yersinia CO-92 Biovar NO DATA [26 34 35 21] [23 23 19 22] pestis Orientalis Yersinia KIM5 P12 NO DATA [26 34 35 21] [23 23 19 22] pestis (Biovar Mediaevalis) Yersinia 91001 NO DATA [26 34 35 21] [23 23 19 22] pestis Haemophilus KW20 NO DATA NO DATA NO DATA influenzae Pseudomonas PAO1 NO DATA NO DATA NO DATA aeruginosa Pseudomonas Pf0-1 NO DATA NO DATA NO DATA fluorescens Pseudomonas KT2440 NO DATA [21 37 37 NO DATA putida 21] Legionella Philadelphia-1 NO DATA NO DATA NO DATA pneumophila Francisella schu 4 NO DATA NO DATA NO DATA tularensis Bordetella Tohama I NO DATA NO DATA NO DATA pertussis Burkholderia J2315 NO DATA NO DATA NO DATA cepacia Burkholderia K96243 NO DATA NO DATA NO DATA pseudomallei Neisseria FA 1090, ATCC NO DATA NO DATA NO DATA gonorrhoeae 700825 Neisseria MC58 (serogroup B) NO DATA NO DATA NO DATA meningitidis Neisseria serogroup C, NO DATA NO DATA NO DATA meningitidis FAM18 Neisseria Z2491 NO DATA NO DATA NO DATA meningitidis (serogroup A) Chlamydophila TW-183 NO DATA NO DATA NO DATA pneumoniae Chlamydophila AR39 NO DATA NO DATA NO DATA pneumoniae Chlamydophila CWL029 NO DATA NO DATA NO DATA pneumoniae Chlamydophila J138 NO DATA NO DATA NO DATA pneumoniae Corynebacterium NCTC13129 NO DATA NO DATA NO DATA diphtheriae Mycobacterium k10 NO DATA NO DATA NO DATA avium Mycobacterium 104 NO DATA NO DATA NO DATA avium Mycobacterium CSU#93 NO DATA NO DATA NO DATA tuberculosis Mycobacterium CDC 1551 NO DATA NO DATA NO DATA tuberculosis Mycobacterium H37Rv (lab NO DATA NO DATA NO DATA tuberculosis strain) Mycoplasma M129 NO DATA NO DATA NO DATA pneumoniae Staphylococcus MRSA252 NO DATA NO DATA NO DATA aureus Staphylococcus MSSA476 NO DATA NO DATA NO DATA aureus Staphylococcus COL NO DATA NO DATA NO DATA aureus Staphylococcus Mu50 NO DATA NO DATA NO DATA aureus Staphylococcus MW2 NO DATA NO DATA NO DATA aureus Staphylococcus N315 NO DATA NO DATA NO DATA aureus Staphylococcus NCTC 8325 NO DATA NO DATA NO DATA aureus Streptococcus NEM316 NO DATA NO DATA NO DATA agalactiae Streptococcus NC_002955 NO DATA NO DATA NO DATA equi Streptococcus MGAS8232 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS315 NO DATA NO DATA NO DATA pyogenes Streptococcus SSI-1 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS10394 NO DATA NO DATA NO DATA pyogenes Streptococcus Manfredo (M5) NO DATA NO DATA NO DATA pyogenes Streptococcus SF370 (M1) NO DATA NO DATA NO DATA pyogenes Streptococcus 670 NO DATA NO DATA NO DATA pneumoniae Streptococcus R6 NO DATA NO DATA NO DATA pneumoniae Streptococcus TIGR4 NO DATA NO DATA NO DATA pneumoniae Streptococcus NCTC7868 NO DATA NO DATA NO DATA gordonii Streptococcus NCTC 12261 NO DATA NO DATA NO DATA mitis Streptococcus UA159 NO DATA NO DATA NO DATA mutans

TABLE 7E Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 362, 363, and 367 Primer 362 Primer 363 Primer 367 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella MGH78578 [21 33 22 [16 34 26 NO DATA pneumoniae 16] 26] Yersinia CO-92 Biovar [20 34 18 NO DATA NO DATA pestis Orientalis 20] Yersinia KIM5 P12 [20 34 18 NO DATA NO DATA pestis (Biovar Mediaevalis) 20] Yersinia pestis 91001 [20 34 18 20] NO DATA NO DATA Haemophilus KW20 NO DATA NO DATA NO DATA influenzae Pseudomonas PAO1 [19 35 21 [16 36 28 NO DATA aeruginosa 17] 22] Pseudomonas Pf0-1 NO DATA [18 35 26 NO DATA fluorescens 23] Pseudomonas KT2440 NO DATA [16 35 28 NO DATA putida 23] Legionella Philadelphia-1 NO DATA NO DATA NO DATA pneumophila Francisella schu 4 NO DATA NO DATA NO DATA tularensis Bordetella Tohama I [20 31 24 [15 34 32 [26 25 34 pertussis 17] 21] 19] Burkholderia J2315 [20 33 21 [15 36 26 [25 27 32 cepacia 18] 25] 20] Burkholderia K96243 [19 34 19 [15 37 28 [25 27 32 pseudomallei 20] 22] 20] Neisseria FA 1090, ATCC NO DATA NO DATA NO DATA gonorrhoeae 700825 Neisseria MC58 (serogroup NO DATA NO DATA NO DATA meningitidis B) Neisseria serogroup C, NO DATA NO DATA NO DATA meningitidis FAM18 Neisseria Z2491 NO DATA NO DATA NO DATA meningitidis (serogroup A) Chlamydophila TW-183 NO DATA NO DATA NO DATA pneumoniae Chlamydophila AR39 NO DATA NO DATA NO DATA pneumoniae Chlamydophila CWL029 NO DATA NO DATA NO DATA pneumoniae Chlamydophila J138 NO DATA NO DATA NO DATA pneumoniae Corynebacterium NCTC13129 NO DATA NO DATA NO DATA diphtheriae Mycobacterium k10 [19 34 23 NO DATA [24 26 35 avium 16] 19] Mycobacterium 104 [19 34 23 NO DATA [24 26 35 avium 16] 19] Mycobacterium CSU#93 [19 31 25 NO DATA [25 25 34 tuberculosis 17] 20] Mycobacterium CDC 1551 [19 31 24 NO DATA [25 25 34 tuberculosis 18] 20] Mycobacterium H37Rv (lab [19 31 24 NO DATA [25 25 34 tuberculosis strain) 18] 20] Mycoplasma M129 NO DATA NO DATA NO DATA pneumoniae Staphylococcus MRSA252 NO DATA NO DATA NO DATA aureus Staphylococcus MSSA476 NO DATA NO DATA NO DATA aureus Staphylococcus COL NO DATA NO DATA NO DATA aureus Staphylococcus Mu50 NO DATA NO DATA NO DATA aureus Staphylococcus MW2 NO DATA NO DATA NO DATA aureus Staphylococcus N315 NO DATA NO DATA NO DATA aureus Staphylococcus NCTC 8325 NO DATA NO DATA NO DATA aureus Streptococcus NEM316 NO DATA NO DATA NO DATA agalactiae Streptococcus NC_002955 NO DATA NO DATA NO DATA equi Streptococcus MGAS8232 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS315 NO DATA NO DATA NO DATA pyogenes Streptococcus SSI-1 NO DATA NO DATA NO DATA pyogenes Streptococcus MGAS10394 NO DATA NO DATA NO DATA pyogenes Streptococcus Manfredo (M5) NO DATA NO DATA NO DATA pyogenes Streptococcus SF370 (M1) NO DATA NO DATA NO DATA pyogenes Streptococcus 670 NO DATA NO DATA NO DATA pneumoniae Streptococcus R6 [20 30 19 NO DATA NO DATA pneumoniae 23] Streptococcus TIGR4 [20 30 19 NO DATA NO DATA pneumoniae 23] Streptococcus NCTC7868 NO DATA NO DATA NO DATA gordonii Streptococcus NCTC 12261 NO DATA NO DATA NO DATA mitis Streptococcus UA159 NO DATA NO DATA NO DATA mutans

Four sets of throat samples from military recruits at different military facilities taken at different time points were analyzed using selected primers disclosed herein. The first set was collected at a military training center from Nov. 1 to Dec. 20, 2002 during one of the most severe outbreaks of pneumonia associated with group A Streptococcus in the United States since 1968. During this outbreak, fifty-one throat swabs were taken from both healthy and hospitalized recruits and plated on blood agar for selection of putative group A Streptococcus colonies. A second set of 15 original patient specimens was taken during the height of this group A Streptococcus-associated respiratory disease outbreak. The third set were historical samples, including twenty-seven isolates of group A Streptococcus, from disease outbreaks at this and other military training facilities during previous years. The fourth set of samples was collected from five geographically separated military facilities in the continental U.S. in the winter immediately following the severe November/December 2002 outbreak.

Pure colonies isolated from group A Streptococcus-selective media from all four collection periods were analyzed with the surveillance primer set. All samples showed base compositions that precisely matched the four completely sequenced strains of Streptococcus pyogenes. Shown in FIG. 4 is a 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.

In addition to the identification of Streptococcus pyogenes, other potentially pathogenic organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid was amplified by primer pair number 349 (SEQ ID NOs: 401:1156) exhibited signals of bioagent identifying amplicons with molecular masses that were found to correspond to analogous base compositions of bioagent identifying amplicons of Streptococcus pyogenes (A27 G32 C24 T18), Neisseria meningitidis (A25 G27 C22 T18), and Haemophilus influenzae (A28 G28 C25 T20) (see FIG. 5 and Table 7B). These organisms were present in a ratio of 4:5:20 as determined by comparison of peak heights with peak height of an internal PCR calibration standard as described in commonly owned PCT Publication Number WO 2005/098047 which is incorporated herein by reference in its entirety.

Since certain division-wide primers that target housekeeping genes are designed to provide coverage of specific divisions of bacteria to increase the confidence level for identification of bacterial species, they are not expected to yield bioagent identifying amplicons for organisms outside of the specific divisions. For example, primer pair number 356 (SEQ ID NOs: 449:1380) primarily amplifies the nucleic acid of members of the classes Bacilli and Clostridia and is not expected to amplify proteobacteria such as Neisseria meningitidis and Haemophilus influenzae. As expected, analysis of the mass spectrum of amplification products obtained with primer pair number 356 does not indicate the presence of Neisseria meningitidis and Haemophilus influenzae but does indicate the presence of Streptococcus pyogenes (FIGS. 3 and 6, Table 7B). Thus, these primers or types of primers can confirm the absence of particular bioagents from a sample.

The 15 throat swabs from military recruits were found to contain a relatively small set of microbes in high abundance. The most common were Haemophilus influenza, Neisseria meningitides, and Streptococcus pyogenes. Staphylococcus epidermidis, Moraxella catarrhalis, Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus were present in fewer samples. An equal number of samples from healthy volunteers from three different geographic locations, were identically analyzed. Results indicated that the healthy volunteers have bacterial flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the viridans group streptococci (S. parasangunis, S. vestibularis, S. mitis, S. oralis and S. pneumoniae; data not shown), and none of the organisms found in the military recruits were found in the healthy controls at concentrations detectable by mass spectrometry. Thus, the military recruits in the midst of a respiratory disease outbreak had a dramatically different microbial population than that experienced by the general population in the absence of epidemic disease.

Example 7 Triangulation Genotyping Analysis for Determination of emm-Type of Streptococcus pyogenes in Epidemic Surveillance

As a continuation of the epidemic surveillance investigation of Example 6, determination of sub-species characteristics (genotyping) of Streptococcus pyogenes, was carried out based on a strategy that generates strain-specific signatures according to the rationale of Multi-Locus Sequence Typing (MLST). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced (Enright et al. Infection and Immunity, 2001, 69, 2416-2427). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced. In the present investigation, bioagent identifying amplicons from housekeeping genes were produced using drill-down primers and analyzed by mass spectrometry. Since mass spectral analysis results in molecular mass, from which base composition can be determined, the challenge was to determine whether resolution of emm classification of strains of Streptococcus pyogenes could be determined.

For the purpose of development of a triangulation genotyping assay, an alignment was constructed of concatenated alleles of seven MLST housekeeping genes (glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS), xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyl transferase (yqiL)) from each of the 212 previously emm-typed strains of Streptococcus pyogenes. From this alignment, the number and location of primer pairs that would maximize strain identification via base composition was determined. As a result, 6 primer pairs were chosen as standard drill-down primers for determination of emm-type of Streptococcus pyogenes. These six primer pairs are displayed in Table 8. This drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

TABLE 8 Triangulation Genotyping Analysis Primer Pairs for Group A Streptococcus Drill-Down Forward Reverse Primer Primer Primer Pair Forward Primer (SEQ ID Reverse Primer (SEQ ID Target No. Name NO:) Name NO:) Gene 442 SP101_SPET11_358_387_TMOD_F 588 SP101_SPET11_448_473_TMOD_R 998 gki 80 SP101_SPET11_358_387_F 126 SP101_SPET11_448_473_TMOD_R 766 gki 443 SP101_SPET11_600_629_TMOD_F 348 SP101_SPET11_686_714_TMOD_R 1018 gtr 81 SP101_SPET11_600_629_F 62 SP101_SPET11_686_714_R 772 gtr 426 SP101_SPET11_1314_1336_TMOD_F 363 SP101_SPET11_1403_1431_TMOD_R 849 murI 86 SP101_SPET11_1314_1336_F 68 SP101_SPET11_1403_1431_R 711 murI 430 SP101_SPET11_1807_1835_TMOD_F 235 SP101_SPET11_1901_1927_TMOD_R 1439 mutS 90 SP101_SPET11_1807_1835_F 33 SP101_SPET11_1901_1927_R 1412 mutS 438 SP101_SPET11_3075_3103_TMOD_F 473 SP101_SPET11_3168_3196_TMOD_R 875 xpt 96 SP101_SPET11_3075_3103_F 108 SP101_SPET11_3168_3196_R 715 xpt 441 SP101_SPET11_3511_3535_TMOD_F 531 SP101_SPET11_3605_3629_TMOD_R 1294 yqiL 98 SP101_SPET11_3511_3535_F 116 SP101_SPET11_3605_3629_R 832 yqiL

The primers of Table 8 were used to produce bioagent identifying amplicons from nucleic acid present in the clinical samples. The bioagent identifying amplicons which were subsequently analyzed by mass spectrometry and base compositions corresponding to the molecular masses were calculated.

Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 9A-C rows 1-3), all except three samples were found to represent emm3, a Group A Streptococcus genotype previously associated with high respiratory virulence. The three outliers were from samples obtained from healthy individuals and probably represent non-epidemic strains. Archived samples (Tables 9A-C rows 5-13) from historical collections showed a greater heterogeneity of base compositions and emm types as would be expected from different epidemics occurring at different places and dates. The results of the mass spectrometry analysis and emm gene sequencing were found to be concordant for the epidemic and historical samples.

TABLE 9A Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 426 and 430 murI mutS emm-type emm- (Primer (Primer # of by Mass Gene Location Pair No. Pair No. Instances Spectrometry Sequencing (sample) Year 426) 430) 48  3 3 MCRD 2002 A39 G25 C20 A38 G27 C23 San T34 T33 2 6 6 Diego A40 G24 C20 A38 G27 C23 (Cultured) T34 T33 1 28  28  A39 G25 C20 A38 G27 C23 T34 T33 15  3 ND A39 G25 C20 A38 G27 C23 T34 T33 6 3 3 NHRC 2003 A39 G25 C20 A38 G27 C23 San T34 T33 3 5, 58 5 Diego- A40 G24 C20 A38 G27 C23 Archive T34 T33 6 6 6 (Cultured) A40 G24 C20 A38 G27 C23 T34 T33 1 11  11  A39 G25 C20 A38 G27 C23 T34 T33 3 12  12  A40 G24 C20 A38 G26 C24 T34 T33 1 22  22  A39 G25 C20 A38 G27 C23 T34 T33 3 25, 75 75  A39 G25 C20 A38 G27 C23 T34 T33 4 44/61, 82, 9 44/61 A40 G24 C20 A38 G26 C24 T34 T33 2 53, 91 91  A39 G25 C20 A38 G27 C23 T34 T33 1 2 2 Ft. 2003 A39 G25 C20 A38 G27 C24 Leonard T34 T32 2 3 3 Wood A39 G25 C20 A38 G27 C23 (Cultured) T34 T33 1 4 4 A39 G25 C20 A38 G27 C23 T34 T33 1 6 6 A40 G24 C20 A38 G27 C23 T34 T33 11  25 or 75 75  A39 G25 C20 A38 G27 C23 T34 T33 1 25, 75, 75  A39 G25 C20 A38 G27 C23 33, T34 T33 34, 4, 52, 84 1 44/61 or 44/61 A40 G24 C20 A38 G26 C24 82 or 9 T34 T33 2 5 or 58 5 A40 G24 C20 A38 G27 C23 T34 T33 3 1 1 Ft. 2003 A40 G24 C20 A38 G27 C23 Sill T34 T33 2 3 3 (Cultured) A39 G25 C20 A38 G27 C23 T34 T33 1 4 4 A39 G25 C20 A38 G27 C23 T34 T33 1 28  28  A39 G25 C20 A38 G27 C23 T34 T33 1 3 3 Ft. 2003 A39 G25 C20 A38 G27 C23 Benning T34 T33 1 4 4 (Cultured) A39 G25 C20 A38 G27 C23 T34 T33 3 6 6 A40 G24 C20 A38 G27 C23 T34 T33 1 11  11  A39 G25 C20 A38 G27 C23 T34 T33 1 13   94** A40 G24 C20 A38 G27 C23 T34 T33 1 44/61 or 82  A40 G24 C20 A38 G26 C24 82 or 9 T34 T33 1 5 or 58 58  A40 G24 C20 A38 G27 C23 T34 T33 1 78 or 89 89  A39 G25 C20 A38 G27 C23 T34 T33 2 5 or 58 ND Lackland 2003 A40 G24 C20 A38 G27 C23 AFB T34 T33 1 2 (Throat A39 G25 C20 A38 G27 C24 Swabs) T34 T32 1 81 or 90 A40 G24 C20 A38 G27 C23 T34 T33 1 78  A38 G26 C20 A38 G27 C23 T34 T33   3*** No No No detection detection detection 7 3 ND MCRD 2002 A39 G25 C20 A38 G27 C23 San T34 T33 1 3 ND Diego No A38 G27 C23 (Throat detection T33 1 3 ND Swabs) No No detection detection 1 3 ND No No detection detection 2 3 ND No A38 G27 C23 detection T33 3 No ND No No detection detection detection

TABLE 9B Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 xpt yqiL emm-type emm- (Primer (Primer # of by Mass Gene Location Pair No. Pair No. Instances Spectrometry Sequencing (sample) Year 438) 441) 48  3 3 MCRD 2002 A30 G36 C20 A40 G29 C19 San T36 T31 2 6 6 Diego A30 G36 C20 A40 G29 C19 (Cultured) T36 T31 1 28  28  A30 G36 C20 A41 G28 C18 T36 T32 15  3 ND A30 G36 C20 A40 G29 C19 T36 T31 6 3 3 NHRC 2003 A30 G36 C20 A40 G29 C19 San T36 T31 3 5, 58 5 Diego- A30 G36 C20 A40 G29 C19 Archive T36 T31 6 6 6 (Cultured) A30 G36 C20 A40 G29 C19 T36 T31 1 11  11  A30 G36 C20 A40 G29 C19 T36 T31 3 12  12  A30 G36 C19 A40 G29 C19 T37 T31 1 22  22  A30 G36 C20 A40 G29 C19 T36 T31 3 25, 75 75  A30 G36 C20 A40 G29 C19 T36 T31 4 44/61, 82, 9 44/61 A30 G36 C20 A41 G28 C19 T36 T31 2 53, 91 91  A30 G36 C19 A40 G29 C19 T37 T31 1 2 2 Ft. 2003 A30 G36 C20 A40 G29 C19 Leonard T36 T31 2 3 3 Wood A30 G36 C20 A40 G29 C19 (Cultured) T36 T31 1 4 4 A30 G36 C19 A41 G28 C19 T37 T31 1 6 6 A30 G36 C20 A40 G29 C19 T36 T31 11  25 or 75 75  A30 G36 C20 A40 G29 C19 T36 T31 1 25, 75, 75  A30 G36 C19 A40 G29 C19 33, T37 T31 34, 4, 52, 84 1 44/61 or 44/61 A30 G36 C20 A41 G28 C19 82 or 9 T36 T31 2 5 or 58 5 A30 G36 C20 A40 G29 C19 T36 T31 3 1 1 Ft. 2003 A30 G36 C19 A40 G29 C19 Sill T37 T31 2 3 3 (Cultured) A30 G36 C20 A40 G29 C19 T36 T31 1 4 4 A30 G36 C19 A41 G28 C19 T37 T31 1 28  28  A30 G36 C20 A41 G28 C18 T36 T32 1 3 3 Ft. 2003 A30 G36 C20 A40 G29 C19 Benning T36 T31 1 4 4 (Cultured) A30 G36 C19 A41 G28 C19 T37 T31 3 6 6 A30 G36 C20 A40 G29 C19 T36 T31 1 11  11  A30 G36 C20 A40 G29 C19 T36 T31 1 13   94** A30 G36 C20 A41 G28 C19 T36 T31 1 44/61 or 82  A30 G36 C20 A41 G28 C19 82 or 9 T36 T31 1 5 or 58 58  A30 G36 C20 A40 G29 C19 T36 T31 1 78 or 89 89  A30 G36 C20 A41 G28 C19 T36 T31 2 5 or 58 ND Lackland 2003 A30 G36 C20 A40 G29 C19 AFB T36 T31 1 2 (Throat A30 G36 C20 A40 G29 C19 Swabs) T36 T31 1 81 or 90 A30 G36 C20 A40 G29 C19 T36 T31 1 78  A30 G36 C20 A41 G28 C19 T36 T31   3*** No No No detection detection detection 7 3 ND MCRD 2002 A30 G36 C20 A40 G29 C19 San T36 T31 1 3 ND Diego A30 G36 C20 A40 G29 C19 (Throat T36 T31 1 3 ND Swabs) A30 G36 C20 No T36 detection 1 3 ND No A40 G29 C19 detection T31 2 3 ND A30 G36 C20 A40 G29 C19 T36 T31 3 No ND No No detection detection detection

TABLE 9C Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 gki gtr emm-type emm- (Primer ((Primer # of by Mass Gene Location Pair No. Pair No. Instances Spectrometry Sequencing (sample) Year 442) 443) 48  3 3 MCRD 2002 A32 G35 C17 A39 G28 C16 San T32 T32 2 6 6 Diego A31 G35 C17 A39 G28 C15 (Cultured) T33 T33 1 28  28  A30 G36 C17 A39 G28 C16 T33 T32 15  3 ND A32 G35 C17 A39 G28 C16 T32 T32 6 3 3 NHRC 2003 A32 G35 C17 A39 G28 C16 San T32 T32 3 5, 58 5 Diego- A30 G36 C20 A39 G28 C15 Archive T30 T33 6 6 6 (Cultured) A31 G35 C17 A39 G28 C15 T33 T33 1 11  11  A30 G36 C20 A39 G28 C16 T30 T32 3 12  12  A31 G35 C17 A39 G28 C15 T33 T33 1 22  22  A31 G35 C17 A38 G29 C15 T33 T33 3 25, 75 75  A30 G36 C17 A39 G28 C15 T33 T33 4 44/61, 82, 9 44/61 A30 G36 C18 A39 G28 C15 T32 T33 2 53, 91 91  A32 G35 C17 A39 G28 C16 T32 T32 1 2 2 Ft. 2003 A30 G36 C17 A39 G28 C15 Leonard T33 T33 2 3 3 Wood A32 G35 C17 A39 G28 C16 (Cultured) T32 T32 1 4 4 A31 G35 C17 A39 G28 C15 T33 T33 1 6 6 A31 G35 C17 A39 G28 C15 T33 T33 11  25 or 75 75  A30 G36 C17 A39 G28 C15 T33 T33 1 25, 75, 75  A30 G36 C17 A39 G28 C15 33, T33 T33 34, 4, 52, 84 1 44/61 or 44/61 A30 G36 C18 A39 G28 C15 82 or 9 T32 T33 2 5 or 58 5 A30 G36 C20 A39 G28 C15 T30 T33 3 1 1 Ft. 2003 A30 G36 C18 A39 G28 C15 Sill T32 T33 2 3 3 (Cultured) A32 G35 C17 A39 G28 C16 T32 T32 1 4 4 A31 G35 C17 A39 G28 C15 T33 T33 1 28  28  A30 G36 C17 A39 G28 C16 T33 T32 1 3 3 Ft. 2003 A32 G35 C17 A39 G28 C16 Benning T32 T32 1 4 4 (Cultured) A31 G35 C17 A39 G28 C15 T33 T33 3 6 6 A31 G35 C17 A39 G28 C15 T33 T33 1 11  11  A30 G36 C20 A39 G28 C16 T30 T32 1 13   94** A30 G36 C19 A39 G28 C15 T31 T33 1 44/61 or 82  A30 G36 C18 A39 G28 C15 82 or 9 T32 T33 1 5 or 58 58  A30 G36 C20 A39 G28 C15 T30 T33 1 78 or 89 89  A30 G36 C18 A39 G28 C15 T32 T33 2 5 or 58 ND Lackland 2003 A30 G36 C20 A39 G28 C15 AFB T30 T33 1 2 (Throat A30 G36 C17 A39 G28 C15 Swabs) T33 T33 1 81 or 90 A30 G36 C17 A39 G28 C15 T33 T33 1 78  A30 G36 C18 A39 G28 C15 T32 T33   3*** No No No detection detection detection 7 3 ND MCRD 2002 A32 G35 C17 A39 G28 C16 San T32 T32 1 3 ND Diego No No (Throat detection detection 1 3 ND Swabs) A32 G35 C17 A39 G28 C16 T32 T32 1 3 ND A32 G35 C17 No T32 detection 2 3 ND A32 G35 C17 No T32 detection 3 No ND No No detection detection detection

Example 8 Design of Calibrant Polynucleotides Based on Bioagent Identifying Amplicons for Identification of Species of Bacteria (Bacterial Bioagent Identifying Amplicons)

This example describes the design of 19 calibrant polynucleotides based on bacterial bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table 5) and the Bacillus anthracis drill-down set (Table 6).

Calibration sequences were designed to simulate bacterial bioagent identifying amplicons produced by the T modified primer pairs shown in Tables 5 and 6 (primer names have the designation “TMOD”). The calibration sequences were chosen as a representative member of the section of bacterial genome from specific bacterial species which would be amplified by a given primer pair. The model bacterial species upon which the calibration sequences are based are also shown in Table 10. For example, the calibration sequence chosen to correspond to an amplicon produced by primer pair no. 361 is SEQ ID NO: 1445. In Table 10, the forward (_F) or reverse (_R) primer name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes e.g.: the forward primer name 16S_EC_(—)713_(—)732_TMOD_F indicates that the forward primer hybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence (in this case, the reference sequence is an extraction consisting of residues 4033120-4034661 of the genomic sequence of E. coli K12 (GenBank gi number 16127994). Additional gene coordinate reference information is shown in Table 11. The designation “TMOD” in the primer names indicates that the 5′ end of the primer has been modified with a non-matched template T residue which prevents the PCR polymerase from adding non-templated adenosine residues to the 5′ end of the amplification product, an occurrence which may result in miscalculation of base composition from molecular mass data (vide supra).

The 19 calibration sequences described in Tables 10 and 11 were combined into a single calibration polynucleotide sequence (SEQ ID NO: 1464—which is herein designated a “combination calibration polynucleotide”) which was then cloned into a pCR®-Blunt vector (Invitrogen, Carlsbad, Calif.). This combination calibration polynucleotide can be used in conjunction with the primers of Tables 5 or 6 as an internal standard to produce calibration amplicons for use in determination of the quantity of any bacterial bioagent. Thus, for example, when the combination calibration polynucleotide vector is present in an amplification reaction mixture, a calibration amplicon based on primer pair 346 (16S rRNA) will be produced in an amplification reaction with primer pair 346 and a calibration amplicon based on primer pair 363 (rpoC) will be produced with primer pair 363. Coordinates of each of the 19 calibration sequences within the calibration polynucleotide (SEQ ID NO: 1464) are indicated in Table 11.

TABLE 10 Bacterial Primer Pairs for Production of Bacterial Bioagent Identifying Amplicons and Corresponding Representative Calibration Sequences Forward Reverse Primer Primer Calibration Calibration Primer (SEQ (SEQ Sequence Sequence Pair Forward Primer ID Reverse Primer ID Model (SEQ ID No. Name NO:) Name NO:) Species NO:) 361 16S_EC_1090_1111_2_TMOD_F 697 16S_EC_1175_1196_TMOD_R 1398 Bacillus 1445 anthracis 346 16S_EC_713_732_TMOD_F 202 16S_EC_789_809_TMOD_R 1110 Bacillus 1446 anthracis 347 16S_EC_785_806_TMOD_F 560 16S_EC_880_897_TMOD_R 1278 Bacillus 1447 anthracis 348 16S_EC_960_981_TMOD_F 706 16S_EC_1054_1073_TMOD_R 895 Bacillus 1448 anthracis 349 23S_EC_1826_1843_TMOD_F 401 23S_EC_1906_1924_TMOD_R 1156 Bacillus 1449 anthracis 360 23S_EC_2646_2667_TMOD_F 409 23S_EC_2745_2765_TMOD_R 1434 Bacillus 1450 anthracis 350 CAPC_BA_274_303_TMOD_F 476 CAPC_BA_349_376_TMOD_R 1314 Bacillus 1451 anthracis 351 CYA_BA_1353_1379_TMOD_F 355 CYA_BA_1448_1467_TMOD_R 1423 Bacillus 1452 anthracis 352 INFB_EC_1365_1393_TMOD_F 687 INFB_EC_1439_1467_TMOD_R 1411 Bacillus 1453 anthracis 353 LEF_BA_756_781_TMOD_F 220 LEF_BA_843_872_TMOD_R 1394 Bacillus 1454 anthracis 356 RPLB_EC_650_679_TMOD_F 449 RPLB_EC_739_762_TMOD_R 1380 Clostridium 1455 botulinum 449 RPLB_EC_690_710_F 309 RPLB_EC_737_758_R 1336 Clostridium 1456 botulinum 359 RPOB_EC_1845_1866_TMOD_F 659 RPOB_EC_1909_1929_TMOD_R 1250 Yersinia 1457 Pestis 362 RPOB_EC_3799_3821_TMOD_F 581 RPOB_EC_3862_3888_TMOD_R 1325 Burkholderia 1458 mallei 363 RPOC_EC_2146_2174_TMOD_F 284 RPOC_EC_2227_2245_TMOD_R 898 Burkholderia 1459 mallei 354 RPOC_EC_2218_2241_TMOD_F 405 RPOC_EC_2313_2337_TMOD_R 1072 Bacillus 1460 anthracis 355 SSPE_BA_115_137_TMOD_F 255 SSPE_BA_197_222_TMOD_R 1402 Bacillus 1461 anthracis 367 TUFB_EC_957_979_TMOD_F 308 TUFB_EC_1034_1058_TMOD_R 1276 Burkholderia 1462 mallei 358 VALS_EC_1105_1124_TMOD_F 385 VALS_EC_1195_1218_TMOD_R 1093 Yersinia 1463 Pestis

TABLE 11 Primer Pair Gene Coordinate References and Calibration Polynucleotide Sequence Coordinates within the Combination Calibration Polynucleotide Coordinates of Reference Calibration Sequence Gene Extraction GenBank GI No. of in Combination Bacterial Coordinates Genomic (G) or Primer Calibration Gene and of Genomic or Plasmid (P) Pair Polynucleotide (SEQ Species Plasmid Sequence Sequence No. ID NO: 1464) 16S E. coli 4033120 . . . 4034661 16127994 (G) 346  16 . . . 109 16S E. coli 4033120 . . . 4034661 16127994 (G) 347  83 . . . 190 16S E. coli 4033120 . . . 4034661 16127994 (G) 348 246 . . . 353 16S E. coli 4033120 . . . 4034661 16127994 (G) 361 368 . . . 469 23S E. coli 4166220 . . . 4169123 16127994 (G) 349 743 . . . 837 23S E. coli 4166220 . . . 4169123 16127994 (G) 360 865 . . . 981 rpoB E. coli. 4178823 . . . 4182851 16127994 (G) 359 1591 . . . 1672 (complement strand) rpoB E. coli 4178823 . . . 4182851 16127994 (G) 362 2081 . . . 2167 (complement strand) rpoC E. coli 4182928 . . . 4187151 16127994 (G) 354 1810 . . . 1926 rpoC E. coli 4182928 . . . 4187151 16127994 (G) 363 2183 . . . 2279 infB E. coli 3313655 . . . 3310983 16127994 (G) 352 1692 . . . 1791 (complement strand) tufB E. coli 4173523 . . . 4174707 16127994 (G) 367 2400 . . . 2498 rplB E. coli 3449001 . . . 3448180 16127994 (G) 356 1945 . . . 2060 rplB E. coli 3449001 . . . 3448180 16127994 (G) 449 1986 . . . 2055 valS E. coli 4481405 . . . 4478550 16127994 (G) 358 1462 . . . 1572 (complement strand) capC 56074 . . . 55628  6470151 (P) 350 2517 . . . 2616 B. anthracis (complement strand) cya 156626 . . . 154288  4894216 (P) 351 1338 . . . 1449 B. anthracis (complement strand) lef 127442 . . . 129921  4894216 (P) 353 1121 . . . 1234 B. anthracis sspE 226496 . . . 226783 30253828 (G) 355 1007-1104 B. anthracis

Example 9 Use of a Calibration Polynucleotide for Determining the Quantity of Bacillus Anthracis in a Sample Containing a Mixture of Microbes

The process described in this example is shown in FIG. 2. The capC gene is a gene involved in capsule synthesis which resides on the pX02 plasmid of Bacillus anthracis. Primer pair number 350 (see Tables 10 and 11) was designed to identify Bacillus anthracis via production of a bacterial bioagent identifying amplicon. Known quantities of the combination calibration polynucleotide vector described in Example 8 were added to amplification mixtures containing bacterial bioagent nucleic acid from a mixture of microbes which included the Ames strain of Bacillus anthracis. Upon amplification of the bacterial bioagent nucleic acid and the combination calibration polynucleotide vector with primer pair no. 350, bacterial bioagent identifying amplicons and calibration amplicons were obtained and characterized by mass spectrometry. A mass spectrum measured for the amplification reaction is shown in FIG. 7. The molecular masses of the bioagent identifying amplicons provided the means for identification of the bioagent from which they were obtained (Ames strain of Bacillus anthracis) and the molecular masses of the calibration amplicons provided the means for their identification as well. The relationship between the abundance (peak height) of the calibration amplicon signals and the bacterial bioagent identifying amplicon signals provides the means of calculation of the copies of the pX02 plasmid of the Ames strain of Bacillus anthracis. Methods of calculating quantities of molecules based on internal calibration procedures are well known to those of ordinary skill in the art.

Averaging the results of 10 repetitions of the experiment described above, enabled a calculation that indicated that the quantity of Ames strain of Bacillus anthracis present in the sample corresponds to approximately 10 copies of pX02 plasmid.

Example 10 Triangulation Genotyping Analysis of Campylobacter Species

A series of triangulation genotyping analysis primers were designed as described in Example 1 with the objective of identification of different strains of Campylobacter jejuni. The primers are listed in Table 12 with the designation “CJST_CJ.” Housekeeping genes to which the primers hybridize and produce bioagent identifying amplicons include: tkt (transketolase), glyA (serine hydroxymethyltransferase), gltA (citrate synthase), aspA (aspartate ammonia lyase), glnA (glutamine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).

TABLE 12 Campylobacter Genotyping Primer Pairs Reverse Primer Forward Primer Pair Forward Primer Primer Reverse Primer (SEQ ID Target No. Name (SEQ ID NO:) Name NO:) Gene 1053 CJST_CJ_1080_1110_F 681 CJST_CJ_1166_1198_R 1022 gltA 1047 CJST_CJ_584_616_F 315 CJST_CJ_663_692_R 1379 glnA 1048 CJST_CJ_360_394_F 346 CJST_CJ_442_476_R 955 aspA 1049 CJST_CJ_2636_2668_F 504 CJST_CJ_2753_2777_R 1409 tkt 1054 CJST_CJ_2060_2090_F 323 CJST_CJ_2148_2174_R 1068 pgm 1064 CJST_CJ_1680_1713_F 479 CJST_CJ_1795_1822_R 938 glyA

The primers were used to amplify nucleic acid from 50 food product samples provided by the USDA, 25 of which contained Campylobacter jejuni and 25 of which contained Campylobacter coli. Primers used in this study were developed primarily for the discrimination of Campylobacter jejuni clonal complexes and for distinguishing Campylobacter jejuni from Campylobacter coli. Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates demonstrate polymorphisms between strains. The conclusions of the comparison of base composition analysis with sequence analysis are shown in Tables 13A-C.

TABLE 13A Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down MLST Primer Pair Nos: 1048 and 1047 Base Base Composition Composition MLST of Bioagent of Bioagent MLST type Type or Identifying Identifying or Clonal Clonal Amplicon Amplicon Complex by Complex Obtained Obtained with Base by with Primer Primer Pair Isolate Composition Sequence Pair No: 1048 No: 1047 Group Species origin analysis analysis Strain (aspA) (glnA) J-1 C. jejuni Goose ST 690/ ST 991 RM3673 A30 G25 A47 G21 692/707/ C16 T46 C16 T25 991 J-2 C. jejuni Human Complex ST RM4192 A30 G25 A48 G21 206/48/353 356, C16 T46 C17 T23 complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A30 G25 A48 G21 354/179 C15 T47 C18 T22 J-4 C. jejuni Human Complex ST RM4197 A30 G25 A48 G21 257 257, C16 T46 C18 T22 complex 257 J-5 C. jejuni Human Complex ST 52, RM4277 A30 G25 A48 G21 52 complex C16 T46 C17 T23 52 J-6 C. jejuni Human Complex ST 51, RM4275 A30 G25 A48 G21 443 complex C15 T47 C17 T23 443 RM4279 A30 G25 A48 G21 C15 T47 C17 T23 J-7 C. jejuni Human Complex ST RM1864 A30 G25 A48 G21 42 604, C15 T47 C18 T22 complex 42 J-8 C. jejuni Human Complex ST RM3193 A30 G25 A48 G21 42/49/362 362, C15 T47 C18 T22 complex 362 J-9 C. jejuni Human Complex ST RM3203 A30 G25 A47 G21 45/283 147, C15 T47 C18 T23 Complex 45 C. jejuni Human Consistent ST 828 RM4183 A31 G27 A48 G21 with C20 T39 C16 T24 C-1 C. coli 74 ST 832 RM1169 A31 G27 A48 G21 closely C20 T39 C16 T24 related ST RM1857 A31 G27 A48 G21 sequence 1056 C20 T39 C16 T24 Poultry types ST 889 RM1166 A31 G27 A48 G21 (none C20 T39 C16 T24 belong ST 829 RM1182 A31 G27 A48 G21 to a C20 T39 C16 T24 clonal ST RM1518 A31 G27 A48 G21 complex) 1050 C20 T39 C16 T24 ST RM1521 A31 G27 A48 G21 1051 C20 T39 C16 T24 ST RM1523 A31 G27 A48 G21 1053 C20 T39 C16 T24 ST RM1527 A31 G27 A48 G21 1055 C20 T39 C16 T24 ST RM1529 A31 G27 A48 G21 1017 C20 T39 C16 T24 ST 860 RM1840 A31 G27 A48 G21 C20 T39 C16 T24 ST RM2219 A31 G27 A48 G21 1063 C20 T39 C16 T24 ST RM2241 A31 G27 A48 G21 1066 C20 T39 C16 T24 ST RM2243 A31 G27 A48 G21 1067 C20 T39 C16 T24 ST RM2439 A31 G27 A48 G21 1068 C20 T39 C16 T24 Swine ST RM3230 A31 G27 A48 G21 1016 C20 T39 C16 T24 ST RM3231 A31 G27 A48 G21 1069 C20 T39 C16 T24 ST RM1904 A31 G27 A48 G21 1061 C20 T39 C16 T24 Unknown ST 825 RM1534 A31 G27 A48 G21 C20 T39 C16 T24 ST 901 RM1505 A31 G27 A48 G21 C20 T39 C16 T24 C-2 C. coli Human ST 895 ST 895 RM1532 A31 G27 A48 G21 C19 T40 C16 T24 C-3 C. coli Poultry Consistent ST RM2223 A31 G27 A48 G21 with 1064 C20 T39 C16 T24 63 ST RM1178 A31 G27 A48 G21 closely 1082 C20 T39 C16 T24 related ST RM1525 A31 G27 A48 G21 sequence 1054 C20 T39 C16 T24 types ST RM1517 A31 G27 A48 G21 (none 1049 C20 T39 C16 T24 Marmoset belong ST 891 RM1531 A31 G27 A48 G21 to a C20 T39 C16 T24 clonal complex)

TABLE 13B Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down MLST Primer Pair Nos: 1053 and 1064 Base Base Composition Composition of Bioagent of Bioagent MLST Identifying Identifying MLST type Type or Amplicon Amplicon or Clonal Clonal Obtained Obtained Complex by Complex with Primer with Primer Base by Pair Pair Isolate Composition Sequence No: 1053 No: 1064 Group Species origin analysis analysis Strain (gltA) (glyA) J-1 C. jejuni Goose ST 690/ ST 991 RM3673 A24 G25 A40 G29 692/707/ C23 T47 C29 T45 991 J-2 C. jejuni Human Complex ST RM4192 A24 G25 A40 G29 206/48/353 356, C23 T47 C29 T45 complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A24 G25 A40 G29 354/179 C23 T47 C29 T45 J-4 C. jejuni Human Complex ST RM4197 A24 G25 A40 G29 257 257, C23 T47 C29 T45 complex 257 J-5 C. jejuni Human Complex ST 52, RM4277 A24 G25 A39 G30 52 complex C23 T47 C26 T48 52 J-6 C. jejuni Human Complex ST 51, RM4275 A24 G25 A39 G30 443 complex C23 T47 C28 T46 443 RM4279 A24 G25 A39 G30 C23 T47 C28 T46 J-7 C. jejuni Human Complex ST RM1864 A24 G25 A39 G30 42 604, C23 T47 C26 T48 complex 42 J-8 C. jejuni Human Complex ST RM3193 A24 G25 A38 G31 42/49/362 362, C23 T47 C28 T46 complex 362 J-9 C. jejuni Human Complex ST RM3203 A24 G25 A38 G31 45/283 147, C23 T47 C28 T46 Complex 45 C. jejuni Human Consistent ST 828 RM4183 A23 G24 A39 G30 with C26 T46 C27 T47 C-1 C. coli 74 ST 832 RM1169 A23 G24 A39 G30 closely C26 T46 C27 T47 related ST RM1857 A23 G24 A39 G30 sequence 1056 C26 T46 C27 T47 Poultry types ST 889 RM1166 A23 G24 A39 G30 (none C26 T46 C27 T47 belong ST 829 RM1182 A23 G24 A39 G30 to a C26 T46 C27 T47 clonal ST RM1518 A23 G24 A39 G30 complex) 1050 C26 T46 C27 T47 ST RM1521 A23 G24 A39 G30 1051 C26 T46 C27 T47 ST RM1523 A23 G24 A39 G30 1053 C26 T46 C27 T47 ST RM1527 A23 G24 A39 G30 1055 C26 T46 C27 T47 ST RM1529 A23 G24 A39 G30 1017 C26 T46 C27 T47 ST 860 RM1840 A23 G24 A39 G30 C26 T46 C27 T47 ST RM2219 A23 G24 A39 G30 1063 C26 T46 C27 T47 ST RM2241 A23 G24 A39 G30 1066 C26 T46 C27 T47 ST RM2243 A23 G24 A39 G30 1067 C26 T46 C27 T47 ST RM2439 A23 G24 A39 G30 1068 C26 T46 C27 T47 Swine ST RM3230 A23 G24 A39 G30 1016 C26 T46 C27 T47 ST RM3231 A23 G24 NO DATA 1069 C26 T46 ST RM1904 A23 G24 A39 G30 1061 C26 T46 C27 T47 Unknown ST 825 RM1534 A23 G24 A39 G30 C26 T46 C27 T47 ST 901 RM1505 A23 G24 A39 G30 C26 T46 C27 T47 C-2 C. coli Human ST 895 ST 895 RM1532 A23 G24 A39 G30 C26 T46 C27 T47 C-3 C. coli Poultry Consistent ST RM2223 A23 G24 A39 G30 with 1064 C26 T46 C27 T47 63 ST RM1178 A23 G24 A39 G30 closely 1082 C26 T46 C27 T47 related ST RM1525 A23 G24 A39 G30 sequence 1054 C25 T47 C27 T47 types ST RM1517 A23 G24 A39 G30 (none 1049 C26 T46 C27 T47 Marmoset belong ST 891 RM1531 A23 G24 A39 G30 to a C26 T46 C27 T47 clonal complex)

TABLE 13C Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down MLST Primer Pair Nos: 1054 and 1049 Base Base Composition Composition MLST of Bioagent of Bioagent MLST type Type or Identifying Identifying or Clonal Clonal Amplicon Amplicon Complex by Complex Obtained Obtained Base by with Primer with Primer Isolate Composition Sequence Pair No: 1054 Pair Group Species origin analysis analysis Strain (pgm) No: 1049 (tkt) J-1 C. jejuni Goose ST 690/ ST 991 RM3673 A26 G33 A41 G28 692/707/ C18 T38 C35 T38 991 J-2 C. jejuni Human Complex ST RM4192 A26 G33 A41 G28 206/48/353 356, C19 T37 C36 T37 complex 353 J-3 C. jejuni Human Complex ST 436 RM4194 A27 G32 A42 G28 354/179 C19 T37 C36 T36 J-4 C. jejuni Human Complex ST RM4197 A27 G32 A41 G29 257 257, C19 T37 C35 T37 complex 257 J-5 C. jejuni Human Complex ST 52, RM4277 A26 G33 A41 G28 52 complex C18 T38 C36 T37 52 J-6 C. jejuni Human Complex ST 51, RM4275 A27 G31 A41 G28 443 complex C19 T38 C36 T37 443 RM4279 A27 G31 A41 G28 C19 T38 C36 T37 J-7 C. jejuni Human Complex ST RM1864 A27 G32 A42 G28 42 604, C19 T37 C35 T37 complex 42 J-8 C. jejuni Human Complex ST RM3193 A26 G33 A42 G28 42/49/362 362, C19 T37 C35 T37 complex 362 J-9 C. jejuni Human Complex ST RM3203 A28 G31 A43 G28 45/283 147, C19 T37 C36 T35 Complex 45 C. jejuni Human Consistent ST 828 RM4183 A27 G30 A46 G28 with C19 T39 C32 T36 C-1 C. coli 74 ST 832 RM1169 A27 G30 A46 G28 closely C19 T39 C32 T36 related ST RM1857 A27 G30 A46 G28 sequence 1056 C19 T39 C32 T36 Poultry types ST 889 RM1166 A27 G30 A46 G28 (none C19 T39 C32 T36 belong ST 829 RM1182 A27 G30 A46 G28 to a C19 T39 C32 T36 clonal ST RM1518 A27 G30 A46 G28 complex) 1050 C19 T39 C32 T36 ST RM1521 A27 G30 A46 G28 1051 C19 T39 C32 T36 ST RM1523 A27 G30 A46 G28 1053 C19 T39 C32 T36 ST RM1527 A27 G30 A46 G28 1055 C19 T39 C32 T36 ST RM1529 A27 G30 A46 G28 1017 C19 T39 C32 T36 ST 860 RM1840 A27 G30 A46 G28 C19 T39 C32 T36 ST RM2219 A27 G30 A46 G28 1063 C19 T39 C32 T36 ST RM2241 A27 G30 A46 G28 1066 C19 T39 C32 T36 ST RM2243 A27 G30 A46 G28 1067 C19 T39 C32 T36 ST RM2439 A27 G30 A46 G28 1068 C19 T39 C32 T36 Swine ST RM3230 A27 G30 A46 G28 1016 C19 T39 C32 T36 ST RM3231 A27 G30 A46 G28 1069 C19 T39 C32 T36 ST RM1904 A27 G30 A46 G28 1061 C19 T39 C32 T36 Unknown ST 825 RM1534 A27 G30 A46 G28 C19 T39 C32 T36 ST 901 RM1505 A27 G30 A46 G28 C19 T39 C32 T36 C-2 C. coli Human ST 895 ST 895 RM1532 A27 G30 A45 G29 C19 T39 C32 T36 C-3 C. coli Poultry Consistent ST RM2223 A27 G30 A45 G29 with 1064 C19 T39 C32 T36 63 ST RM1178 A27 G30 A45 G29 closely 1082 C19 T39 C32 T36 related ST RM1525 A27 G30 A45 G29 sequence 1054 C19 T39 C32 T36 types ST RM1517 A27 G30 A45 G29 (none 1049 C19 T39 C32 T36 Marmoset belong ST 891 RM1531 A27 G30 A45 G29 to a C19 T39 C32 T36 clonal complex)

The base composition analysis method was successful in identification of 12 different strain groups. Campylobacter jejuni and Campylobacter coli are generally differentiated by all loci. Ten clearly differentiated Campylobacter jejuni isolates and 2 major Campylobacter coli groups were identified even though the primers were designed for strain typing of Campylobacter jejuni. One isolate (RM4183) which was designated as Campylobacter jejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli by full MLST sequencing.

Example 11 Identification of Acinetobacter baumannii Using Broad Range Survey and Division-Wide Primers in Epidemiological Surveillance

To test the capability of the broad range survey and division-wide primer sets of Table 5 in identification of Acinetobacter species, 183 clinical samples were obtained from individuals participating in, or in contact with individuals participating in Operation Iraqi Freedom (including US service personnel, US civilian patients at the Walter Reed Army Institute of Research (WRAIR), medical staff, Iraqi civilians and enemy prisoners. In addition, 34 environmental samples were obtained from hospitals in Iraq, Kuwait, Germany, the United States and the USNS Comfort, a hospital ship.

Upon amplification of nucleic acid obtained from the clinical samples, primer pairs 346-349, 360, 361, 354, 362 and 363 (Table 5) all produced bacterial bioagent amplicons which identified Acinetobacter baumannii in 215 of 217 samples. The organism Klebsiella pneumoniae was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain of Acinetobacter baumannii) were identified and assigned arbitrary numbers from 1 to 14. Strain type 1 was found in 134 of the sample isolates and strains 3 and 7 were found in 46 and 9 of the isolates respectively.

The epidemiology of strain type 7 of Acinetobacter baumannii was investigated. Strain 7 was found in 4 patients and 5 environmental samples (from field hospitals in Iraq and Kuwait). The index patient infected with strain 7 was a pre-war patient who had a traumatic amputation in March of 2003 and was treated at a Kuwaiti hospital. The patient was subsequently transferred to a hospital in Germany and then to WRAIR. Two other patients from Kuwait infected with strain 7 were found to be non-infectious and were not further monitored. The fourth patient was diagnosed with a strain 7 infection in September of 2003 at WRAIR. Since the fourth patient was not related involved in Operation Iraqi Freedom, it was inferred that the fourth patient was the subject of a nosocomial infection acquired at WRAIR as a result of the spread of strain 7 from the index patient.

The epidemiology of strain type 3 of Acinetobacter baumannii was also investigated. Strain type 3 was found in 46 samples, all of which were from patients (US service members, Iraqi civilians and enemy prisoners) who were treated on the USNS Comfort hospital ship and subsequently returned to Iraq or Kuwait. The occurrence of strain type 3 in a single locale may provide evidence that at least some of the infections at that locale were a result of nosocomial infections.

This example thus illustrates an embodiment wherein the methods of analysis of bacterial bioagent identifying amplicons provide the means for epidemiological surveillance.

Example 12 Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Acinetobacter baumanii

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, an additional 21 primer pairs were selected based on analysis of housekeeping genes of the genus Acinetobacter. Genes to which the drill-down triangulation genotyping analysis primers hybridize for production of bacterial bioagent identifying amplicons include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutY), fumarate hydratase (fumC), and pyrophosphate phospho-hydratase (ppa). These 21 primer pairs are indicated with reference to sequence listings in Table 14. Primer pair numbers 1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers 1155-1157 hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers 1165-1170 hybridize to and amplify segments of fumC. Primer pair number 1171 hybridizes to and amplifies a segment of ppa. Primer pair numbers: 2846-2848 hybridize to and amplify segments of the parC gene of DNA topoisomerase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii. Primer pair numbers 2852-2854 hybridize to and amplify segments of the gyrA gene of DNA gyrase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii. Primer pair numbers 2922 and 2972 are speciating primers which are useful for identifying different species members of the genus Acinetobacter. The primer names given in Table 14A (with the exception of primer pair numbers 2846-2848, 2852-2854) indicate the coordinates to which the primers hybridize to a reference sequence which comprises a concatenation of the genes TrpE, efp (elongation factor p), adk, mutT, fumC, and ppa. For example, the forward primer of primer pair 1151 is named AB_MLST-11-OIF007_(—)62_(—)91_F because it hybridizes to the Acinetobacter primer reference sequence of strain type 11 in sample 007 of Operation Iraqi Freedom (OIF) at positions 62 to 91. DNA was sequenced from strain type 11 and from this sequence data and an artificial concatenated sequence of partial gene extractions was assembled for use in design of the triangulation genotyping analysis primers. The stretches of arbitrary residues “N”s in the concatenated sequence were added for the convenience of separation of the partial gene extractions (40N for AB_MLST (SEQ ID NO: 1471)).

The hybridization coordinates of primer pair numbers 2846-2848 are with respect to GenBank Accession number X95819. The hybridization coordinates of primer pair numbers 2852-2854 are with respect to GenBank Accession number AY642140. Sequence residue “I” appearing in the forward and reverse primers of primer pair number 2972 represents inosine.

TABLE 14A Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter Forward Reverse Primer Primer Primer Pair (SEQ ID (SEQ ID No. Forward Primer Name NO:) Reverse Primer Name NO:) 1151 AB_MLST-11- 454 AB_MLST-11- 1418 OIF007_62_91_F OIF007_169_203_R 1152 AB_MLST-11- 243 AB_MLST-11- 969 OIF007_185_214_F OIF007_291_324_R 1153 AB_MLST-11- 541 AB_MLST-11- 1400 OIF007_260_289_F OIF007_364_393_R 1154 AB_MLST-11- 436 AB_MLST-11- 1036 OIF007_206_239_F OIF007_318_344_R 1155 AB_MLST-11- 378 AB_MLST-11- 1392 OIF007_522_552_F OIF007_587_610_R 1156 AB_MLST-11- 250 AB_MLST-11- 902 OIF007_547_571_F OIF007_656_686_R 1157 AB_MLST-11- 256 AB_MLST-11- 881 OIF007_601_627_F OIF007_710_736_R 1158 AB_MLST-11- 384 AB_MLST-11- 878 OIF007_1202_1225_F OIF007_1266_1296_R 1159 AB_MLST-11- 384 AB_MLST-11- 1199 OIF007_1202_1225_F OIF007_1299_1316_R 1160 AB_MLST-11- 694 AB_MLST-11- 1215 OIF007_1234_1264_F OIF007_1335_1362_R 1161 AB_MLST-11- 225 AB_MLST-11- 1212 OIF007_1327_1356_F OIF007_1422_1448_R 1162 AB_MLST-11- 383 AB_MLST-11- 1083 OIF007_1345_1369_F OIF007_1470_1494_R 1163 AB_MLST-11- 662 AB_MLST-11- 1083 OIF007_1351_1375_F OIF007_1470_1494_R 1164 AB_MLST-11- 422 AB_MLST-11- 1083 OIF007_1387_1412_F OIF007_1470_1494_R 1165 AB_MLST-11- 194 AB_MLST-11- 1173 OIF007_1542_1569_F OIF007_1656_1680_R 1166 AB_MLST-11- 684 AB_MLST-11- 1173 OIF007_1566_1593_F OIF007_1656_1680_R 1167 AB_MLST-11- 375 AB_MLST-11- 890 OIF007_1611_1638_F OIF007_1731_1757_R 1168 AB_MLST-11- 182 AB_MLST-11- 1195 OIF007_1726_1752_F OIF007_1790_1821_R 1169 AB_MLST-11- 656 AB_MLST-11- 1151 OIF007_1792_1826_F OIF007_1876_1909_R 1170 AB_MLST-11- 656 AB_MLST-11- 1224 OIF007_1792_1826_F OIF007_1895_1927_R 1171 AB_MLST-11- 618 AB_MLST-11- 1157 OIF007_1970_2002_F OIF007_2097_2118_R 2846 PARC_X95819_33_58_F 302 PARC_X95819_121_153_R 852 2847 PARC_X95819_33_58_F 199 PARC_X95819_157_178_R 889 2848 PARC_X95819_33_58_F 596 PARC_X95819_97_128_R 1169 2852 GYRA_AY642140_−1_24_F 150 GYRA_AY642140_71_100_R 1242 2853 GYRA_AY642140_26_54_F 166 GYRA_AY642140_121_146_R 1069 2854 GYRA_AY642140_26_54_F 166 GYRA_AY642140_58_89_R 1168 2922 AB_MLST-11- 583 AB_MLST-11- 923 OIF007_991_1018_F OIF007_1110_1137_R 2972 AB_MLST-11- 592 AB_MLST-11- 924 OIF007_1007_1034_F OIF007_1126_1153_R

TABLE 14B Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter Primer Forward Reverse Pair Primer Primer No. (SEQ ID NO:) SEQUENCE (SEQ ID NO:) SEQUENCE 1151 454 TGAGATTGCTGAACATTTAATG 1418 TTGTACATTTGAAACAATATGC CTGATTGA ATGACATGTGAAT 1152 243 TATTGTTTCAAATGTACAAGGT 969 TCACAGGTTCTACTTCATCAAT GAAGTGCG AATTTCCATTGC 1153 541 TGGAACGTTATCAGGTGCCCCA 1400 TTGCAATCGACATATCCATTTC AAAATTCG ACCATGCC 1154 436 TGAAGTGCGTGATGATATCGAT 1036 TCCGCCAAAAACTCCCCTTTTC GCACTTGATGTA ACAGG 1155 378 TCGGTTTAGTAAAAGAACGTAT 1392 TTCTGCTTGAGGAATAGTGCGT TGCTCAACC GG 1156 250 TCAACCTGACTGCGTGAATGGT 902 TACGTTCTACGATTTCTTCATC TGT AGGTACATC 1157 256 TCAAGCAGAAGCTTTGGAAGAA 881 TACAACGTGATAAACACGACCA GAAGG GAAGC 1158 384 TCGTGCCCGCAATTTGCATAAA 878 TAATGCCGGGTAGTGCAATCCA GC TTCTTCTAG 1159 384 TCGTGCCCGCAATTTGCATAAA 1199 TGCACCTGCGGTCGAGCG GC 1160 694 TTGTAGCACAGCAAGGCAAATT 1215 TGCCATCCATAATCACGCCATA TCCTGAAAC CTGACG 1161 225 TAGGTTTACGTCAGTATGGCGT 1212 TGCCAGTTTCCACATTTCACGT GATTATGG TCGTG 1162 383 TCGTGATTATGGATGGCAACGT 1083 TCGCTTGAGTGTAGTCATGATT GAA GCG 1163 662 TTATGGATGGCAACGTGAAACG 1083 TCGCTTGAGTGTAGTCATGATT CGT GCG 1164 422 TCTTTGCCATTGAAGATGACTT 1083 TCGCTTGAGTGTAGTCATGATT AAGC GCG 1165 194 TACTAGCGGTAAGCTTAAACAA 1173 TGAGTCGGGTTCACTTTACCTG GATTGC GCA 1166 684 TTGCCAATGATATTCGTTGGTT 1173 TGAGTCGGGTTCACTTTACCTG AGCAAG GCA 1167 375 TCGGCGAAATCCGTATTCCTGA 890 TACCGGAAGCACCAGCGACATT AAATGA AATAG 1168 182 TACCACTATTAATGTCGCTGGT 1195 TGCAACTGAATAGATTGCAGTA GCTTC AGTTATAAGC 1169 656 TTATAACTTACTGCAATCTATT 1151 TGAATTATGCAAGAAGTGATCA CAGTTGCTTGGTG ATTTTCTCACGA 1170 656 TTATAACTTACTGCAATCTATT 1224 TGCCGTAACTAACATAAGAGAA CAGTTGCTTGGTG TTATGCAAGAA 1171 618 TGGTTATGTACCAAATACTTTG 1157 TGACGGCATCGATACCACCGTC TCTGAAGATGG 2846 302 TCCAAAAAAATCAGCGCGTACA 852 TAAAGGATAGCGGTAACTAAAT GTGG GGCTGAGCCAT 2847 199 TACTTGGTAAATACCACCCACA 889 TACCCCAGTTCCCCTGACCTTC TGGTGA 2848 596 TGGTAAATACCACCCACATGGT 1169 TGAGCCATGAGTACCATGGCTT GAC CATAACATGC 2852 150 TAAATCTGCCCGTGTCGTTGGT 1242 TGCTAAAGTCTTGAGCCATACG GAC AACAATGG 2853 166 TAATCGGTAAATATCACCCGCA 1069 TCGATCGAACCGAAGTTACCCT TGGTGAC GACC 2854 166 TAATCGGTAAATATCACCCGCA 1168 TGAGCCATACGAACAATGGTTT TGGTGAC CATAAACAGC 2922 583 TGGGCGATGCTGCGAAATGGTT 923 TAGTATCACCACGTACACCCGG AAAAGA ATCAGT 2972 592 TGGGIGATGCTGCIAAATGGTT 924 TAGTATCACCACGTACICCIGG AAAAGA ATCAGT

Analysis of bioagent identifying amplicons obtained using the primers of Table 14B for over 200 samples from Operation Iraqi Freedom resulted in the identification of 50 distinct strain type clusters. The largest cluster, designated strain type 11 (ST11) includes 42 sample isolates, all of which were obtained from US service personnel and Iraqi civilians treated at the 28^(th) Combat Support Hospital in Baghdad. Several of these individuals were also treated on the hospital ship USNS Comfort. These observations are indicative of significant epidemiological correlation/linkage.

All of the sample isolates were tested against a broad panel of antibiotics to characterize their antibiotic resistance profiles. As an example of a representative result from antibiotic susceptibility testing, ST11 was found to consist of four different clusters of isolates, each with a varying degree of sensitivity/resistance to the various antibiotics tested which included penicillins, extended spectrum penicillins, cephalosporins, carbepenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics. Thus, the genotyping power of bacterial bioagent identifying amplicons, particularly drill-down bacterial bioagent identifying amplicons, has the potential to increase the understanding of the transmission of infections in combat casualties, to identify the source of infection in the environment, to track hospital transmission of nosocomial infections, and to rapidly characterize drug-resistance profiles which enable development of effective infection control measures on a time-scale previously not achievable.

Example 13 Triangulation Genotyping Analysis and Codon Analysis of Acinetobacter baumannii Samples from Two Health Care Facilities

In this investigation, 88 clinical samples were obtained from Walter Reed Hospital and 95 clinical samples were obtained from Northwestern Medical Center. All samples from both healthcare facilities were suspected of containing sub-types of Acinetobacter baumannii, at least some of which were expected to be resistant to quinolone drugs. Each of the 183 samples was analyzed by the methods disclosed herein. DNA was extracted from each of the samples and amplified with eight triangulation genotyping analysis primer pairs represented by primer pair numbers: 1151, 1156, 1158, 1160, 1165, 1167, 1170, and 1171. The DNA was also amplified with speciating primer pair number 2922 and codon analysis primer pair numbers 2846-2848, which were designed to interrogate a codon present in the parC gene, and primer pair numbers 2852-2854, which bracket a codon present in the gyrA gene. The parC and gyrA codon mutations are both responsible for causing drug resistance in Acinetobacter baumannii. During evolution of drug resistant strains, the gyrA mutation usually occurs before the parC mutation. Amplification products were measured by ESI-TOF mass spectrometry as indicated in Example 4. The base compositions of the amplification products were calculated from the average molecular masses of the amplification products and are shown in Tables 15-18. The entries in each of the tables are grouped according to strain type number, which is an arbitrary number assigned to Acinetobacter baumannii strains in the order of observance beginning from the triangulation genotyping analysis OIF genotyping study described in Example 12. For example, strain type 11 which appears in samples from the Walter Reed Hospital is the same strain as the strain type 11 mentioned in Example 12. Ibis# refers to the order in which each sample was analyzed. Isolate refers to the original sample isolate numbering system used at the location from which the samples were obtained (either Walter Reed Hospital or Northwestern Medical Center). ST=strain type. ND=not detected. Base compositions highlighted with bold type indicate that the base composition is a unique base composition for the amplification product obtained with the pair of primers indicated.

TABLE 15A Base Compositions of Amplification Products of 88 A. baumannii Samples Obtained from Walter Reed Hospital and Amplified with Codon Analysis Primer Pairs Targeting the gyrA Gene PP No: 2852 PP No: 2853 PP No: 2854 Species Ibis# Isolate ST gyrA gyrA gyrA A. baumannii 20 1082 1 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 13 854 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 22 1162 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 27 1230 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 31 1367 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 37 1459 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 55 1700 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 64 1777 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 73 1861 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 74 1877 10 ND A29G28C21T43 A17G13C13T21 A. baumannii 86 1972 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 3 684 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 6 720 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 7 726 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 19 1079 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 21 1123 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 23 1188 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 33 1417 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 34 1431 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 38 1496 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 40 1523 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 42 1640 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 50 1666 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 51 1668 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 52 1695 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 65 1781 11 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 44 1649 12 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 49A 1658.1 12 A25G23C22T31 A29G28C21T43 A17G13C13T21 A. baumannii 49B 1658.2 12 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 56 1707 12 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 80 1893 12 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 5 693 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 8 749 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 10 839 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 14 865 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 16 888 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 29 1326 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 35 1440 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 41 1524 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 46 1652 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 47 1653 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 48 1657 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 57 1709 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 61 1727 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 63 1762 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 67 1806 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 75 1881 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 77 1886 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 1 649 46 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 2 653 46 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 39 1497 16 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 24 1198 15 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 28 1243 15 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 43 1648 15 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 62 1746 15 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 4 689 15 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 68 1822 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 69 1823A 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 70 1823B 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 71 1826 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 72 1860 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 81 1924 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 82 1929 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 85 1966 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 11 841 3 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 32 1415 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 45 1651 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 54 1697 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 58 1712 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 60 1725 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 66 1802 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 76 1883 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 78 1891 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 79 1892 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 83 1947 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 84 1964 24 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 53 1696 24 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 36 1458 49 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 59 1716 9 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 9 805 30 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 18 967 39 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 30 1322 48 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. baumannii 26 1218 50 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. sp. 13TU 15 875 A1 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. sp. 13TU 17 895 A1 A25G23C22T31 A29G28C22T42 A17G13C14T20 A. sp. 3 12 853 B7 A25G22C22T32 A30G29C22T40 A17G13C14T20 A. johnsonii 25 1202 NEW1 A25G22C22T32 A30G29C22T40 A17G13C14T20 A. sp. 2082 87 2082 NEW2 A25G22C22T32 A31G28C22T40 A17G13C14T20

TABLE 15B Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter Reed Hospital and Amplified with Codon Analysis Primer Pairs Targeting the parC Gene PP No: 2846 PP No: 2847 PP No: 2848 Species Ibis# Isolate ST parC parC parC A. baumannii 20 1082 1 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 13 854 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 22 1162 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 27 1230 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 31 1367 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 37 1459 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 55 1700 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 64 1777 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 73 1861 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 74 1877 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 86 1972 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 3 684 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 6 720 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 7 726 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 19 1079 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 21 1123 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 23 1188 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 33 1417 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 34 1431 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 38 1496 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 40 1523 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 42 1640 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 50 1666 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 51 1668 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 52 1695 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 65 1781 11 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 44 1649 12 A33G26C28T34 A29028C25T32 A16G14C14T16 A. baumannii 49A 1658.1 12 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 49B 1658.2 12 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 56 1707 12 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 80 1893 12 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 5 693 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 8 749 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 10 839 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 14 865 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 16 888 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 29 1326 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 35 1440 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 41 1524 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 46 1652 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 47 1653 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 48 1657 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 57 1709 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 61 1727 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 63 1762 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 67 1806 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 75 1881 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 77 1886 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 1 649 46 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 2 653 46 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 39 1497 16 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 24 1198 15 A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 28 1243 15 A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 43 1648 15 A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 62 1746 15 A33G26C28T34 A29G29C23T33 A16G14C14T16 A. baumannii 4 689 15 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 68 1822 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 69 1823A 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 70 1823B 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 71 1826 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 72 1860 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 81 1924 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 82 1929 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 85 1966 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 11 841 3 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 32 1415 24 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 45 1651 24 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 54 1697 24 A33G26C29T33 A29G28C26T31 A16G14C1ST15 A. baumannii 58 1712 24 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 60 1725 24 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 66 1802 24 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 76 1883 24 A33G26C29T33 A29G28C26T31 A16G14C1ST15 A. baumannii 78 1891 24 A34G25C29T33 A30G27C26T31 A16G14C1ST15 A. baumannii 79 1892 24 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 83 1947 24 A34G25C29T33 A30G27C26T31 A16G14C1ST15 A. baumannii 84 1964 24 A33G26C29T33 A29G28C26T31 A16G14C1ST15 A. baumannii 53 1696 24 A33G26C29T33 A29G28C26T31 A16G14C1ST15 A. baumannii 36 1458 49 A34G26C29T32 A30G28C24T32 A16G14C15T15 A. baumannii 59 1716 9 A33G26C29T33 A29G28C26T31 A16G14C1ST15 A. baumannii 9 805 30 A33G26C29T33 A29G28C26T31 A16G14C1ST15 A. baumannii 18 967 39 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 30 1322 48 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 26 1218 50 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. sp. 13TU 15 875 A1 A32G26C28T35 A28G28C24T34 A16G14C15T15 A. sp. 13TU 17 895 A1 A32G26C28T35 A28G28C24T34 A16G14C15T15 A. sp. 3 12 853 B7 A29G26C27T39 A26G32C21T35 A16G14C15T15 A. johnsonii 25 1202 NEW1 A32G28C26T35 A29G29C22T34 A16G14C15T15 A. sp. 2082 87 2082 NEW2 A33G27C26T35 A31G28C20T35 A16G14C15T15

TABLE 16A Base Compositions Determined from A. baumannii DNA Samples Obtained from Northwestem Medical Center and Amplified with Codon Analysis Primer Pairs Targeting the gyrA Gene PP No: 2852 PP No: 2853 PP No: 2854 Species Ibis# Isolate ST gyrA gyrA gyrA A. baumannii 54 536 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 87 665 3 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 8 80 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 9 91 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 10 92 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 11 131 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 12 137 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 21 218 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 26 242 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 94 678 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 1 9 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 2 13 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 3 19 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 4 24 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 5 36 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 6 39 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 13 139 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 15 165 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 16 170 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 17 186 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 20 202 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 22 221 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 24 234 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 25 239 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 33 370 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 34 389 10 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 19 201 14 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 27 257 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 29 301 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 31 354 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 36 422 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 37 424 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 38 434 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 39 473 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 40 482 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 44 512 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 45 516 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 47 522 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 48 526 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 50 528 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 52 531 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 53 533 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 56 542 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 59 550 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 62 556 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 64 557 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 70 588 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 73 603 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 74 605 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 75 606 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 77 611 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 79 622 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 83 643 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 85 653 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 89 669 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 93 674 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 23 228 51 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 32 369 52 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 35 393 52 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 30 339 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 41 485 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 42 493 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 43 502 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 46 520 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 49 527 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 51 529 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 65 562 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 68 579 53 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 57 546 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 58 548 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 60 552 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 61 555 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 63 557 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 66 570 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 67 578 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 69 584 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 71 593 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 72 602 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 76 609 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 78 621 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 80 625 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 81 628 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 82 632 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 84 649 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 86 655 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 88 668 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 90 671 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 91 672 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 92 673 54 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 18 196 55 A25G23C22T31 A29G28C21T43 A17G13C13T21 A. baumannii 55 537 27 A25G23C21T32 A29G28C21T43 A17G13C13T21 A. baumannii 28 263 27 A25G23C22T31 A29G28C22T42 A17013C14T20 A. sp. 3 14 164 B7 A25G22C22T32 A30G29C22T40 A17G13C14T20 mixture 7 71 — ND ND A17G13C15T19

TABLE 16B Base Compositions Determined from A. baumannii DNA Samples Obtained from Northwestem Medical Center and Amplified with Codon Analysis Primer Pairs Targeting the parC Gene PP No: 2846 PP No: 2847 PP No: 2848 Species Ibis# Isolate ST parC parC parC A. baumannii 54 536 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 87 665 3 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 8 80 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 9 91 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 10 92 10 A33G26C28T34 A29G28C25T32 ND A. baumannii 11 131 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 12 137 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 21 218 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 26 242 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 94 678 10 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 1 9 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 2 13 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 3 19 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 4 24 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 5 36 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 6 39 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 13 139 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 15 165 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 16 170 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 17 186 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 20 202 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 22 221 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 24 234 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 25 239 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 33 370 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 34 389 10 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 19 201 14 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 27 257 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 29 301 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 31 354 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 36 422 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 37 424 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 38 434 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 39 473 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 40 482 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 44 512 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 45 516 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 47 522 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 48 526 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 50 528 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 52 531 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 53 533 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 56 542 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumaunii 59 550 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 62 556 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 64 557 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 70 588 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 73 603 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 74 605 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 75 606 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 77 611 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 79 622 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 83 643 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 85 653 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 89 669 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 93 674 51 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 23 228 51 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 32 369 52 A34G25C28T34 A30G27C25T32 A16G14C14T16 A. baumannii 35 393 52 A34G25C28T34 A30G27C25T32 A16G14C14T16 A. baumannii 30 339 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 41 485 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 42 493 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 43 502 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 46 520 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 49 527 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 51 529 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 65 562 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 68 579 53 A34G25C29T33 A30G27C26T31 A16G14C15T15 A. baumannii 57 546 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 58 548 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 60 552 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 61 555 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 63 557 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 66 570 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 67 578 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 69 584 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 71 593 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 72 602 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 76 609 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 78 621 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 80 625 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 81 628 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 82 632 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 84 649 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 86 655 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 88 668 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 90 671 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 91 672 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 92 673 54 A33G26C28T34 A29G28C25T32 A16G14C14T16 A. baumannii 18 196 55 A33G27C28T33 A29G28C25T31 A15G14C15T16 A. baumannii 55 537 27 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. baumannii 28 263 27 A33G26C29T33 A29G28C26T31 A16G14C15T15 A. sp. 3 14 164 B7 A35G25C29T32 A30G28C17T39 A16G14C15T15 mixture 7 71 — ND ND A17G14C15T14

TABLE 17A Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter Reed Hospital and Amplified with Speciating Primer Pair No. 2922 and Triangulation Genotyping Analysis Primer Pair Nos. 1151 and 1156 PP No: 2922 PP No: 1151 PP No: 1156 Species Ibis# Isolate ST efp trpE Adk A. baumannii 20 1082 1 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 13 854 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 22 1162 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 27 1230 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 31 1367 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 37 1459 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 55 1700 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 64 1777 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 73 1861 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 74 1877 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 86 1972 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 3 684 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 6 720 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 7 726 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 19 1079 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 21 1123 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 23 1188 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 33 1417 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 34 1431 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 38 1496 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 40 1523 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 42 1640 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 50 1666 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 51 1668 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 52 1695 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 65 1781 11 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 44 1649 12 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 49A 1658.1 12 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 49B 1658.2 12 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 56 1707 12 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 80 1893 12 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 5 693 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 8 749 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 10 839 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 14 865 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 16 888 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 29 1326 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 35 1440 14 A44G35C25T43 ND A44G32C27T37 A. baumannii 41 1524 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 46 1652 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 47 1653 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 48 1657 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 57 1709 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 61 1727 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 63 1762 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 67 1806 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 75 1881 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 77 1886 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 1 649 46 A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 2 653 46 A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 39 1497 16 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 24 1198 15 A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 28 1243 15 A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 43 1648 15 A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 62 1746 15 A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 4 689 15 A44G35C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 68 1822 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 69 1823A 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 70 1823B 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 71 1826 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 72 1860 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 81 1924 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 82 1929 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 85 1966 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 11 841 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 32 1415 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 45 1651 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 54 1697 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 58 1712 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 60 1725 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 66 1802 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 76 1883 24 ND A43G36C20T43 A44G32C27T37 A. baumannii 78 1891 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 79 1892 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 83 1947 24 A44G35C25T43 A43036C20T43 A44G32C27T37 A. baumannii 84 1964 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 53 1696 24 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 36 1458 49 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 59 1716 9 A44G35C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 9 805 30 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 18 967 39 A45G34C25T43 A44G35C22T41 A44G32C26T38 A. baumannii 30 1322 48 A44G35C25T43 A43G36C20T43 A44G32C27T37 A. baumannii 26 1218 50 A44G35C25T43 A44G35C21T42 A44G32C26T38 A. sp. 13TU 15 875 A1 A47G33C24T43 A46G32C20T44 A44G33C27T36 A. sp. 13TU 17 895 A1 A47033C24T43 A46G32C20T44 A44G33C27T36 A. sp. 3 12 853 B7 A46G35C24T42 A42G34C20T46 A43G33C24T40 A. johnsonii 25 1202 NEW1 A46G35C23T43 A42G35C21T44 A43G33C23T41 A. sp. 2082 87 2082 NEW2 A46G36C22T43 A42G32C20T48 A42G34C23T41

TABLE 17B Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter Reed Hospital and Amplified with Triangulation Genotyping Analysis Primer Pair Nos. 1158 and 1160 and 1165 PP No: 1158 PP No: 1160 PP No: 1165 Species Ibis# Isolate ST mutY mutY fumC A. baumannii 20 1082 1 A27G21C25T22 A32G35C29T33 A40G33C30T36 A. baumannii 13 854 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 22 1162 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 27 1230 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 31 1367 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 37 1459 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 55 1700 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 64 1777 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 73 1861 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 74 1877 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 86 1972 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 3 684 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 6 720 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 7 726 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 19 1079 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 21 1123 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 23 1188 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 33 1417 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 34 1431 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 38 1496 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 40 1523 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 42 1640 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 50 1666 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 51 1668 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 52 1695 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 65 1781 11 A27G21C25T22 A32G34C28T35 A40G33C30T36 A. baumannii 44 1649 12 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 49A 1658.1 12 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 49B 1658.2 12 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 56 1707 12 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 80 1893 12 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 5 693 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 8 749 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 10 839 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 14 865 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 16 888 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 29 1326 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 35 1440 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 41 1524 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 46 1652 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 47 1653 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 48 1657 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 57 1709 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 61 1727 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 63 1762 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 67 1806 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 75 1881 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 77 1886 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 1 649 46 A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 2 653 46 A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 39 1497 16 A29G19C26T21 A31G35C29T34 A40G34C29T36 A. baumannii 24 1198 15 A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 28 1243 15 A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 43 1648 15 A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 62 1746 15 A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 4 689 15 A29G19C26T21 A31G35C29T34 A40G33C29T37 A. baumannii 68 1822 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 69 1823A 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 70 1823B 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 71 1826 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 72 1860 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 81 1924 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 82 1929 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 85 1966 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 11 841 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 32 1415 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 45 1651 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 54 1697 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 58 1712 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 60 1725 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 66 1802 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 76 1883 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 78 1891 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 79 1892 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 83 1947 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 84 1964 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 53 1696 24 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 36 1458 49 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 59 1716 9 A27G21C25T22 A32G35C28T34 A39G33C30T37 A. baumannii 9 805 30 A27G21C25T22 A32G35C28T34 A39G33C30T37 A. baumannii 18 967 39 A27G21C26T21 A32G35C28T34 A39G33C30T37 A. baumannii 30 1322 48 A28G21C24T22 A32G35C29T33 A40G33C30T36 A. baumannii 26 1218 50 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. sp. 13TU 15 875 A1 A27G21C25T22 A30G36C26T37 A41G34C28T36 A. sp. 13TU 17 895 A1 A27G21C25T22 A30G36C26T37 A41G34C28T36 A. sp. 3 12 853 B7 A26G23C23T23 A30G36C27T36 A39G37C26T37 A. johnsonii 25 1202 NEW1 A25G23C24T23 A30G35C30T34 A38G37C26T38 A. sp. 2082 87 2082 NEW2 A26G22C24T23 A31G35C28T35 A42G34C27T36

TABLE 17C Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter Reed Hospital and Amplified with Triangulation Genotyping Analysis Primer Pair Nos. 1167 and 1170 and 1171 PP No: 1167 PP No: 1170 PP No: 1171 Species Ibis# Isolate ST fumC fumC ppa A. baumannii 20 1082 1 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 13 854 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 22 1162 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 27 1230 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 31 1367 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 37 1459 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 55 1700 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 64 1777 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 73 1861 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 74 1877 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 86 1972 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 3 684 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 6 720 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 7 726 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 19 1079 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 21 1123 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 23 1188 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 33 1417 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 34 1431 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 38 1496 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 40 1523 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 42 1640 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 50 1666 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 51 1668 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 52 1695 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 65 1781 11 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 44 1649 12 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 49A 1658.1 12 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 49B 1658.2 12 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 56 1707 12 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 80 1893 12 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 5 693 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 8 749 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 10 839 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 14 865 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 16 888 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 29 1326 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 35 1440 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 41 1524 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 46 1652 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 47 1653 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 48 1657 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 57 1709 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 61 1727 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 63 1762 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 67 1806 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 75 1881 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 77 1886 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 1 649 46 A41G35C32T39 A37G28C20T51 A35G37C32T45 A. baumannii 2 653 46 A41G35C32T39 A37G28C20T51 A35G37C32T45 A. baumannii 39 1497 16 A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 24 1198 15 A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 28 1243 15 A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 43 1648 15 A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 62 1746 15 A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 4 689 15 A41G35C32T39 A37G28C20T51 A35G37C30T47 A. baumannii 68 1822 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 69 1823A 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 70 1823B 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 71 1826 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 72 1860 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 81 1924 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 82 1929 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 85 1966 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 11 841 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 32 1415 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 45 1651 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 54 1697 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 58 1712 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 60 1725 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 66 1802 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 76 1883 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 78 1891 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 79 1892 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 83 1947 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 84 1964 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 53 1696 24 A40G35C34T38 A39G26C22T49 A35G37C33T44 A. baumannii 36 1458 49 A40G35C34T38 A39G26C22T49 A35G37C30T47 A. baumannii 59 1716 9 A40G35C32T40 A38G27C20T51 A36G35C31T47 A. baumannii 9 805 30 A40G35C32T40 A38G27C21T50 A35G36C29T49 A. baumannii 18 967 39 A40G35C33T39 A38G27C20T51 A35G37C30T47 A. baumannii 30 1322 48 A40G35C35T37 A38G27C21T50 A35G37C30T47 A. baumannii 26 1218 50 A40G35C34T38 A38G27C21T50 A35G37C33T44 A. sp. 13TU 15 875 A1 A41G39C31T36 A37G26C24T49 A34G38C31T46 A. sp. 13TU 17 895 A1 A41G39C31T36 A37G26C24T49 A34G38C31T46 A. sp. 3 12 853 B7 A43G37C30T37 A36G27C24T49 A34G37C31T47 A. johnsonii 25 1202 NEW1 A42G38C31T36 A40G27C19T50 A35G37C32T45 A. sp. 2082 87 2082 NEW2 A43G37C32T35 A37G26C21T52 A35G38C31T45

TABLE 18A Base Compositions Determined from A. baumannii DNA Samples Obtained from Northwestem Medical Center and Amplified with Speciating Primer Pair No. 2922 and Triangulation Genotyping Analysis Primer Pair Nos. 1151 and 1156 PP No: 2922 PP No: 1151 PP No: 1156 Species Ibis# Isolate ST efp trpE adk A. baumannii 54 536 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 87 665 3 A44G35C24T44 A44G35C22T41 A44G32C26T38 A. baumannii 8 80 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 9 91 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 10 92 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 11 131 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 12 137 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 21 218 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 26 242 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 94 678 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 1 9 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 2 13 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 3 19 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 4 24 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 5 36 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 6 39 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 13 139 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 15 165 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 16 170 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 17 186 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 20 202 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 22 221 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 24 234 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 25 239 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 33 370 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 34 389 10 A45G34C25T43 A44G35C21T42 A44G32C26T38 A. baumannii 19 201 14 A44G35C25T43 A44G35C22T41 A44G32C27T37 A. baumannii 27 257 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 29 301 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 31 354 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 36 422 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 37 424 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 38 434 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 39 473 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 40 482 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 44 512 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 45 516 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 47 522 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 48 526 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 50 528 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 52 531 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 53 533 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 56 542 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 59 550 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 62 556 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 64 557 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 70 588 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 73 603 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 74 605 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 75 606 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 77 611 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 79 622 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 83 643 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 85 653 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 89 669 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 93 674 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 23 228 51 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 32 369 52 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 35 393 52 A44G35C25T43 A43G36C20T43 A44G32C26T38 A. baumannii 30 339 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 41 485 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 42 493 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 43 502 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 46 520 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 49 527 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 51 529 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 65 562 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 68 579 53 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 57 546 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 58 548 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 60 552 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 61 555 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 63 557 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 66 570 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 67 578 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 69 584 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 71 593 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 72 602 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 76 609 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 78 621 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 80 625 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 81 628 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 82 632 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 84 649 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 86 655 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 88 668 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 90 671 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 91 672 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 92 673 54 A44G35C25T43 A44G35C20T43 A44G32C26T38 A. baumannii 18 196 55 A44G35C25T43 A44G35C20T43 A44G32C27T37 A. baumannii 55 537 27 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. baumannii 28 263 27 A44G35C25T43 A44G35C19T44 A44G32C27T37 A. sp. 3 14 164 B7 A46G35C24T42 A42G34C20T46 A43G33C24T40 mixture 7 71 ? mixture ND ND

TABLE 18B Base Compositions Determined from A. baumannii DNA Samples Obtained from Northwestem Medical Center and Amplified with Triangulation Genotyping Analysis Primer Pair Nos. 1158, 1160 and 1165 PP No: 1158 PP No: 1160 PP No: 1165 Species Ibis# Isolate ST mutY mutY fumC A. baumannii 54 536 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 87 665 3 A27G20C27T21 A32G35C28T34 A40G33C30T36 A. baumannii 8 80 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 9 91 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 10 92 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 11 131 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 12 137 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 21 218 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 26 242 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 94 678 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 1 9 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 2 13 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 3 19 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 4 24 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 5 36 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 6 39 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 13 139 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 15 165 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 16 170 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 17 186 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 20 202 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 22 221 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 24 234 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 25 239 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 33 370 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 34 389 10 A27G21C26T21 A32G35C28T34 A40G33C30T36 A. baumannii 19 201 14 A27G21C25T22 A31G36C28T34 A40G33C29T37 A. baumannii 27 257 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 29 301 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 31 354 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 36 422 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 37 424 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 38 434 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 39 473 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 40 482 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 44 512 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 45 516 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 47 522 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 48 526 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 50 528 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 52 531 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 53 533 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 56 542 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 59 550 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 62 556 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 64 557 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 70 588 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 73 603 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 74 605 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 75 606 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 77 611 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 79 622 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 83 643 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 85 653 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 89 669 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 93 674 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 23 228 51 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 32 369 52 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 35 393 52 A27G21C25T22 A32G35C28T34 A40G33C29T37 A. baumannii 30 339 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 41 485 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 42 493 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 43 502 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 46 520 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 49 527 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 51 529 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 65 562 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 68 579 53 A28G20C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 57 546 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 58 548 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 60 552 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 61 555 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 63 557 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 66 570 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 67 578 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 69 584 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 71 593 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 72 602 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 76 609 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 78 621 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 80 625 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 81 628 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 82 632 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 84 649 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 86 655 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 88 668 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 90 671 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 91 672 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 92 673 54 A27G21C26T21 A32G34C29T34 A40G33C30T36 A. baumannii 18 196 55 A27G21C25T22 A31G36C27T35 A40G33C29T37 A. baumannii 55 537 27 A27G21C25T22 A32G35C28T34 A40G33C30T36 A. baumannii 28 263 27 A27G21C25T22 A32G35C28T34 A40G33C30T36 A. sp. 3 14 164 B7 A26G23C23T23 A30G36C27T36 A39G37C26T37 mixture 7 71 ? ND ND ND

TABLE 18C Base Compositions Determined from A. baumannii DNA Samples Obtained from Northwestem Medical Center and Amplified with Triangnlation Genotyping Analysis Primer Pair Nos. 1167, 1170 and 1171 PP No: 1167 PP No: 1170 PP No: 1171 Species Ibis# Isolate ST fumC fumC ppa A. baumannii 54 536 3 A41G34C35T37 A38G27C20T51 A35037C31T46 A. baumannii 87 665 3 A41G34C35T37 A38G27C20T51 A35G37C31T46 A. baumannii 8 80 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 9 91 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 10 92 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 11 131 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 12 137 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 21 218 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 26 242 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 94 678 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 1 9 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 2 13 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 3 19 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 4 24 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 5 36 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 6 39 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 13 139 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 15 165 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 16 170 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 17 186 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 20 202 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 22 221 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 24 234 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 25 239 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 33 370 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 34 389 10 A41G34C34T38 A38G27C21T50 A35G37C33T44 A. baumannii 19 201 14 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 27 257 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 29 301 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 31 354 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 36 422 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 37 424 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 38 434 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 39 473 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 40 482 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 44 512 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 45 516 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 47 522 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 48 526 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 50 528 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 52 531 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 53 533 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 56 542 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 59 550 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 62 556 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 64 557 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 70 588 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 73 603 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 74 605 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 75 606 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 77 611 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 79 622 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 83 643 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 85 653 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 89 669 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 93 674 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 23 228 51 A40G35C34T38 A38G27C21T50 A35G37C30T47 A. baumannii 32 369 52 A40G35C34T38 A38G27C21T50 A35G37C31T46 A. baumannii 35 393 52 A40G35C34T38 A38G27C21T50 A35G37C31T46 A. baumannii 30 339 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 41 485 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 42 493 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 43 502 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 46 520 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 49 527 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 51 529 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 65 562 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 68 579 53 A40G35C35T37 A38G27C21T50 A35G37C31T46 A. baumannii 57 546 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 58 548 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 60 552 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 61 555 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 63 557 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 66 570 54 A40G35C34T38 A39026C22T49 A35G37C31T46 A. baumannii 67 578 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 69 584 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 71 593 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 72 602 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 76 609 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 78 621 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 80 625 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 81 628 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 82 632 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 84 649 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 86 655 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 88 668 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 90 671 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 91 672 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 92 673 54 A40G35C34T38 A39G26C22T49 A35G37C31T46 A. baumannii 18 196 55 A42G34C33T38 A38G27C20T51 A35G37C31T46 A. baumannii 55 537 27 A40G35C33T39 A38G27C20T51 A35G37C33T44 A. baumannii 28 263 27 A40G35C33T39 A38G27C20T51 A35G37C33T44 A. sp. 3 14 164 B7 A43G37C30T37 A36G27C24T49 A34G37C31T47 mixture 7 71 — ND ND ND

Base composition analysis of the samples obtained from Walter Reed hospital indicated that a majority of the strain types identified were the same strain types already characterized by the OIF study of Example 12. This is not surprising since at least some patients from which clinical samples were obtained in OIF were transferred to the Walter Reed Hospital (WRAIR). Examples of these common strain types include: ST10, ST11, ST12, ST14, ST15, ST16 and ST46. A strong correlation was noted between these strain types and the presence of mutations in the gyrA and parC which confer quinolone drug resistance.

In contrast, the results of base composition analysis of samples obtained from Northwestern Medical Center indicate the presence of 4 major strain types: ST10, ST51, ST53 and ST54. All of these strain types have the gyrA quinolone resistance mutation and most also have the parC quinolone resistance mutation, with the exception of ST35. This observation is consistent with the current understanding that the gyrA mutation generally appears before the parC mutation and suggests that the acquisition of these drug resistance mutations is rather recent and that resistant isolates are taking over the wild-type isolates. Another interesting observation was that a single isolate of ST3 (isolate 841) displays a triangulation genotyping analysis pattern similar to other isolates of ST3, but the codon analysis amplification product base compositions indicate that this isolate has not yet undergone the quinolone resistance mutations in gyrA and parC.

The six isolates that represent species other than Acinetobacter baumannii in the samples obtained from the Walter Reed Hospital were each found to not carry the drug resistance mutations.

The results described above involved analysis of 183 samples using the methods and compositions disclosed herein. Results were provided to collaborators at the Walter Reed hospital and Northwestern Medical center within a week of obtaining samples. This example highlights the rapid throughput characteristics of the analysis platform and the resolving power of triangulation genotyping analysis and codon analysis for identification of and determination of drug resistance in bacteria.

Example 14 Identification of Drug Resistance Genes and Virulence Factors in Staphylococcus aureus

An eight primer pair panel was designed for identification of drug resistance genes and virulence factors of Staphylococcus aureus and is shown in Table 19. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 19.

TABLE 19 Primer Pairs for Identification of Drug Resistance Genes and Virulence Factors in Staphylococcus aureus Forward Primer Reverse Primer (SEQ Primer Pair Forward Primer ID Reverse Primer (SEQ ID Target No. Name NO:) Name NO:) Gene  879 MECA_Y14051_4507_4530_F 288 MECA_Y14051_4555_4581_R 1269 mecA 2056 MECI-R_NC003923- 698 MECI-R_NC003923- 1420 MecI-R 41798- 41798- 41609_33_60_F 41609_86_113_R 2081 ERMA_NC002952- 217 ERMA_NC002952- 1167 ermA 55890- 55890- 56621_366_395_F 56621_438_465_R 2086 ERMC_NC005908- 399 ERMC_NC005908- 1041 ermC 2004- 2004- 2738_85_116_F 2738_173_206_R 2095 PVLUK_NC003923- 456 PVLUK_NC003923- 1261 Pv-luk 1529595- 1529595- 1531285_688_713_F 1531285_775_804_R 2249 TUFB_NC002758- 430 TUFB_NC002758- 1321 tufB 615038- 615038- 616222_696_725_F 616222_793_820_R 2256 NUC_NC002758- 174 NUC_NC002758-  853 Nuc 894288- 894288- 894974_316_345_F 894974_396_421_R 2313 MUPR_X75439_2486_2516_F 172 MUPR_X75439_2548_2574_R 1360 mupR

Primer pair numbers 2256 and 2249 are confirmation primers designed with the aim of high level identification of Staphylococcus aureus. The nuc gene is a Staphylococcus aureus-specific marker gene. The tufB gene is a universal housekeeping gene but the bioagent identifying amplicon defined by primer pair number 2249 provides a unique base composition (A43 G28 C19 T35) which distinguishes Staphylococcus aureus from other members of the genus Staphylococcus.

High level methicillin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 879 and 2056. Analyses have indicated that primer pair number 879 is not expected to prime S. sciuri homolog or Enterococcus faecalis/faciem ampicillin-resistant PBP5 homologs.

Macrolide and erythromycin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 2081 and 2086.

Resistance to mupriocin in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2313.

Virulence in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2095. This primer pair can simultaneously and identify the pvl (lukS-PV) gene and the lukD gene which encodes a homologous enterotoxin. A bioagent identifying amplicon of the lukD gene has a six nucleobase length difference relative to the lukS-PV gene.

A total of 32 blinded samples of different strains of Staphylococcus aureus were provided by the Center for Disease Control (CDC). Each sample was analyzed by PCR amplification with the eight primer pair panel, followed by purification and measurement of molecular masses of the amplification products by mass spectrometry. Base compositions for the amplification products were calculated. The base compositions provide the information summarized above for each primer pair. The results are shown in Tables 20A and B. One result noted upon un-blinding of the samples is that each of the PVL+ identifications agreed with PVL+ identified in the same samples by standard PCR assays. These results indicate that the panel of eight primer pairs is useful for identification of drug resistance and virulence sub-species characteristics for Staphylococcus aureus. It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment.

TABLE 20A Drug Resistance and Virulence Identified in Blinded Samples of Various Strains of Staphylococcus aureus with Primer Pair Nos. 2081, 2086, 2095 and 2256 Primer Primer Primer Sample Primer Pair No. Pair No. Pair No. Pair No. Index No. 2081 (ermA) 2086 (ermC) 2095 (pv-luk) 2256 (nuc) CDC0010 − − PVL−/lukD+ + CDC0015 − − PVL+/lukD+ + CDC0019 − + PVL−/lukD+ + CDC0026 + − PVL−/lukD+ + CDC0030 + − PVL−/lukD+ + CDC004 − − PVL+/lukD+ + CDC0014 − + PVL+/lukD+ + CDC008 − − PVL−/lukD+ + CDC001 + − PVL−/lukD+ + CDC0022 + − PVL−/lukD+ + CDC006 + − PVL−/lukD+ + CDC007 − − PVL−/lukD+ + CDCVRSA1 + − PVL−/lukD+ + CDCVRSA2 + + PVL−/lukD+ + CDC0011 + − PVL−/lukD+ + CDC0012 − − PVL+/lukD− + CDC0021 + − PVL−/lukD+ + CDC0023 + − PVL−/lukD+ + CDC0025 + − PVL−/lukD+ + CDC005 − − PVL−/lukD+ + CDC0018 + − PVL+/lukD− + CDC002 − − PVL−/lukD+ + CDC0028 + − PVL−/lukD+ + CDC003 − − PVL−/lukD+ + CDC0013 − − PVL+/lukD+ + CDC0016 − − PVL−/lukD+ + CDC0027 + − PVL−/lukD+ + CDC0029 − − PVL+/lukD+ + CDC0020 − + PVL−/lukD+ + CDC0024 − − PVL−/lukD+ + CDC0031 − − PVL−/lukD+ +

TABLE 20B Drug Resistance and Virulence Identified in Blinded Samples of Various Strains of Staphylococcus aureus with Primer Pair Nos. 2249, 879, 2056, and 2313 Primer Primer Primer Primer Pair No. Sample Pair No. 2249 Pair No. Pair No. 2313 Index No. (tufB) 879 (mecA) 2056 (mecI-R) (mupR) CDC0010 Staphylococcus + + − aureus CDC0015 Staphylococcus − − − aureus CDC0019 Staphylococcus + + − aureus CDC0026 Staphylococcus + + − aureus CDC0030 Staphylococcus + + − aureus CDC004 Staphylococcus + + − aureus CDC0014 Staphylococcus + + − aureus CDC008 Staphylococcus + + − aureus CDC001 Staphylococcus + + − aureus CDC0022 Staphylococcus + + − aureus CDC006 Staphylococcus + + + aureus CDC007 Staphylococcus + + − aureus CDCVRSA1 Staphylococcus + + − aureus CDCVRSA2 Staphylococcus + + − aureus CDC0011 Staphylococcus − − − aureus CDC0012 Staphylococcus + + − aureus CDC0021 Staphylococcus + + − aureus CDC0023 Staphylococcus + + − aureus CDC0025 Staphylococcus + + − aureus CDC005 Staphylococcus + + − aureus CDC0018 Staphylococcus + + − aureus CDC002 Staphylococcus + + − aureus CDC0028 Staphylococcus + + − aureus CDC003 Staphylococcus + + − aureus CDC0013 Staphylococcus + + − aureus CDC0016 Staphylococcus + + − aureus CDC0027 Staphylococcus + + − aureus CDC0029 Staphylococcus + + − aureus CDC0020 Staphylococcus − − − aureus CDC0024 Staphylococcus + + − aureus CDC0031 Staphylococcus − − − scleiferi

Example 15 Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Staphylococcus aureus

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within six different housekeeping genes which are listed in Table 21. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 21.

TABLE 21 Primer Pairs for Triangulation Genotyping Analysis of Staphylococcus aureus Forward Primer Reverse Primer (SEQ Primer Pair ID (SEQ ID Target No. Forward Primer Name NO:) Reverse Primer Name NO:) Gene 2146 ARCC_NC003923- 437 ARCC_NC003923- 1137 arcC 2725050- 2725050- 2724595_131_161_F 2724595_214_245_R 2149 AROE_NC003923- 530 AROE_NC003923- 891 aroE 1674726- 1674726- 1674277_30_62_F 1674277_155_181_R 2150 AROE_NC003923- 474 AROE_NC003923- 869 aroE 1674726- 1674726- 1674277_204_232_F 1674277_308_335_R 2156 GMK_NC003923- 268 GMK_NC003923- 1284 gmk 1190906- 1190906- 1191334_301_329_F 1191334_403_432_R 2157 PTA_NC003923- 418 PTA_NC003923- 1301 pta 628885- 628885- 629355_237_263_F 629355_314_345_R 2161 TPI_NC003923- 318 TPI_NC003923- 1300 tpi 830671- 830671- 831072_1_34_F 831072_97_129_R 2163 YQI_NC003923- 440 YQI_NC003923- 1076 yqi 378916- 378916- 379431_142_167_F 379431_259_284_R 2166 YQI_NC003923- 219 YQI_NC003923- 1013 yqi 378916- 378916- 379431_275_300_F 379431_364_396_R

The same samples analyzed for drug resistance and virulence in Example 14 were subjected to triangulation genotyping analysis. The primer pairs of Table 21 were used to produce amplification products by PCR, which were subsequently purified and measured by mass spectrometry. Base compositions were calculated from the molecular masses and are shown in Tables 22A and 22B.

TABLE 22A Triangulation Genotyping Analysis of Blinded Samples of Various Strains of Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156 Sample Index Primer Pair No. Primer Pair No. Primer Pair No. Primer Pair No. No. Strain 2146 (arcC) 2149(aroE) 2150 (aroE) 2156 (gmk) CDC0010 COL A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC0015 COL A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC0019 COL A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC0026 COL A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC0030 COL A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC004 COL A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC0014 COL A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC008 ???? A44 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T29 T51 T43 T32 CDC001 Mu50 A45 G23 C20 A58 G24 C18 A40 G36 C13 A51 G29 C21 T27 T52 T43 T31 CDC0022 Mu50 A45 G23 C20 A58 G24 C18 A40 G36 C13 A51 G29 C21 T27 T52 T43 T31 CDC006 Mu50 A45 G23 C20 A58 G24 C18 A40 G36 C13 A51 G29 C21 T27 T52 T43 T31 CDC0011 MRSA252 A45 G24 C18 A58 G24 C19 A41 G36 C12 A51 G29 C21 T28 T51 T43 T31 CDC0012 MRSA252 A45 G24 C18 A58 G24 C19 A41 G36 C12 A51 G29 C21 T28 T51 T43 T31 CDC0021 MRSA252 A45 G24 C18 A58 G24 C19 A41 G36 C12 A51 G29 C21 T28 T51 T43 T31 CDC0023 ST:110 A45 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T28 T51 T43 T32 CDC0025 ST:110 A45 G24 C18 A59 G24 C18 A40 G36 C13 A50 G30 C20 T28 T51 T43 T32 CDC005 ST:338 A44 G24 C18 A59 G23 C19 A40 G36 C14 A51 G29 C21 T29 T51 T42 T31 CDC0018 ST:338 A44 G24 C18 A59 G23 C19 A40 G36 C14 A51 G29 C21 T29 T51 T42 T31 CDC002 ST:108 A46 G23 C20 A58 G24 C19 A42 G36 C12 A51 G29 C20 T26 T51 T42 T32 CDC0028 ST:108 A46 G23 C20 A58 G24 C19 A42 G36 C12 A51 G29 C20 T26 T51 T42 T32 CDC003 ST:107 A45 G23 C20 A58 G24 C18 A40 G36 C13 A51 G29 C21 T27 T52 T43 T31 CDC0013 ST:12 ND A59 G24 C18 A40 G36 C13 A51 G29 C21 T51 T43 T31 CDC0016 ST:120 A45 G23 C18 A58 G24 C19 A40 G37 C13 A51 G29 C21 T29 T51 T42 T31 CDC0027 ST:105 A45 G23 C20 A58 G24 C18 A40 G36 C13 A51 G29 C21 T27 T52 T43 T31 CDC0029 MSSA476 A45 G23 C20 A58 G24 C19 A40 G36 C13 A50 G30 C20 T27 T51 T43 T32 CDC0020 ST:15 A44 G23 C21 A59 G23 C18 A40 G36 C13 A50 G30 C20 T27 T52 T43 T32 CDC0024 ST:137 A45 G23 C20 A57 G25 C19 A40 G36 C13 A51 G29 C22 T27 T51 T43 T30 CDC0031 *** No product No product No product No product

TABLE 22B Triangulation Genotyping Analysis of Blinded Samples of Various Strains of Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156 Sample Primer Pair No. Primer Pair No. Primer Pair No. Primer Pair No. Index No. Strain 2157 (pta) 2161 (tpi) 2163 (yqi) 2166 (yqi) CDC0010 COL A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC0015 COL A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC0019 COL A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC0026 COL A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC0030 COL A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC004 COL A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC0014 COL A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC008 unknown A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC001 Mu50 A33 G25 C22 A50 G28 C22 A42 G36 C22 A36 G31 C19 T29 T29 T43 T36 CDC0022 Mu50 A33 G25 C22 A50 G28 C22 A42 G36 C22 A36 G31 C19 T29 T29 T43 T36 CDC006 Mu50 A33 G25 C22 A50 G28 C22 A42 G36 C22 A36 G31 C19 T29 T29 T43 T36 CDC0011 MRSA252 A32 G25 C23 A50 G28 C22 A42 G36 C22 A37 G30 C18 T29 T29 T43 T37 CDC0012 MRSA252 A32 G25 C23 A50 G28 C22 A42 G36 C22 A37 G30 C18 T29 T29 T43 T37 CDC0021 MRSA252 A32 G25 C23 A50 G28 C22 A42 G36 C22 A37 G30 C18 T29 T29 T43 T37 CDC0023 ST:110 A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC0025 ST:110 A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC005 ST:338 A32 G25 C24 A51 G27 C21 A42 G36 C22 A37 G30 C18 T28 T30 T43 T37 CDC0018 ST:338 A32 G25 C24 A51 G27 C21 A42 G36 C22 A37 G30 C18 T28 T30 T43 T37 CDC002 ST:108 A33 G25 C23 A50 G28 C22 A42 G36 C22 A37 G30 C18 T28 T29 T43 T37 CDC0028 ST:108 A33 G25 C23 A50 G28 C22 A42 G36 C22 A37 G30 C18 T28 T29 T43 T37 CDC003 ST:107 A32 G25 C23 A51 G28 C22 A41 G37 C22 A37 G30 C18 T29 T28 T43 T37 CDC0013 ST:12 A32 G25 C23 A51 G28 C22 A42 G36 C22 A37 G30 C18 T29 T28 T43 T37 CDC0016 ST:120 A32 G25 C24 A50 G28 C21 A42 G36 C22 A37 G30 C18 T28 T30 T43 T37 CDC0027 ST:105 A33 G25 C22 A50 G28 C22 A43 G36 C21 A36 G31 C19 T29 T29 T43 T36 CDC0029 MSSA476 A33 G25 C22 A50 G28 C22 A42 G36 C22 A36 G31 C19 T29 T29 T43 T36 CDC0020 ST:15 A33 G25 C22 A50 G28 C21 A42 G36 C22 A36 G31 C18 T29 T30 T43 T37 CDC0024 ST:137 A33 G25 C22 A51 G28 C22 A42 G36 C22 A37 G30 C18 T29 T28 T43 T37 CDC0031 *** A34 G25 C25 A51 G27 C24 No product No product T25 T27 Note: *** The sample CDC0031 was identified as Staphylococcus scleiferi as indicated in Example 14. Thus, the triangulation genotyping primers designed for Staphylococcus aureus would generally not be expected to prime and produce amplification products of this organism. Tables 22A and 22B indicate that amplification products are obtained for this organism only with primer pair numbers 2157 and 2161.

A total of thirteen different genotypes of Staphylococcus aureus were identified according to the unique combinations of base compositions across the eight different bioagent identifying amplicons obtained with the eight primer pairs. These results indicate that this eight primer pair panel is useful for analysis of unknown or newly emerging strains of Staphylococcus aureus. It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment.

Example 16 Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Vibrio

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 23. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 23.

TABLE 23 Primer Pairs for Triangulation Genotyping Analysis of Members of the Bacterial Genus Vibrio Forward Reverse Primer Primer Primer (SEQ (SEQ Pair Forward Primer ID Reverse Primer ID Target No. Name NO:) Name NO:) Gene 1098 RNASEP_VBC_331_349_F 325 RNASEP_VBC_388_414_R 1163 RNAseP 2000 CTXB_NC002505_46_70_F 278 CTXB_NC002505_132_162_R 1039 ctxB 2001 FUR_NC002505_87_113_F 465 FUR_NC002505_205_228_R 1037 fur 2011 GYRB_NC002505_1161_1190_F 148 GYRB_NC002505_1255_1284_R 1172 gyrB 2012 OMPU_NC002505_85_110_F 190 OMPU_NC002505_154_180_R 1254 ompU 2014 OMPU_NC002505_431_455_F 266 OMPU_NC002505_544_567_R 1094 ompU 2323 CTXA_NC002505- 508 CTXA_NC002505- 1297 ctxA 1568114- 1568114- 1567341_122_149_F 1567341_186_214_R 2927 GAPA_NC002505_694_721_F 259 GAPA_NC_002505_29_58_R 1060 gapA

A group of 50 bacterial isolates containing multiple strains of both environmental and clinical isolates of Vibrio cholerae, 9 other Vibrio species, and 3 species of Photobacteria were tested using this panel of primer pairs. Base compositions of amplification products obtained with these 8 primer pairs were used to distinguish amongst various species tested, including sub-species differentiation within Vibrio cholerae isolates. For instance, the non-O1/non-O139 isolates were clearly resolved from the O1 and the O139 isolates, as were several of the environmental isolates of Vibrio cholerae from the clinical isolates.

It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment.

Example 17 Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Pseudomonas

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of twelve triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 24. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 24.

TABLE 24 Primer Pairs for Triangulation Genotyping Analysis of Members of the Bacterial Genus Pseudomonas Forward Primer Reverse Primer (SEQ Primer Pair ID (SEQ ID Target No. Forward Primer Name NO:) Reverse Primer Name NO:) Gene 2949 ACS_NC002516- 376 ACS_NC002516- 1265 acsA 970624- 970624- 971013_299_316_F 971013_364_383_R 2950 ARO_NC002516- 267 ARO_NC002516- 1341 aroE 26883- 26883- 27380_4_26_F 27380_111_128_R 2951 ARO_NC002516- 705 ARO_NC002516- 1056 aroE 26883- 26883- 27380_356_377_F 27380_459_484_R 2954 GUA_NC002516- 710 GUA_NC002516- 1259 guaA 4226546- 4226546- 4226174_155_178_F 4226174_265_287_R 2956 GUA_NC002516- 374 GUA_NC002516- 1111 guaA 4226546- 4226546- 4226174_242_263_F 4226174_355_371_R 2957 MUT_NC002516- 545 MUT_NC002516- 978 mutL 5551158- 5551158- 5550717_5_26_F 5550717_99_116_R 2959 NUO_NC002516- 249 NUO_NC002516- 1095 nuoD 2984589- 2984589- 2984954_8_26_F 2984954_97_117_R 2960 NUO_NC002516- 195 NUO_NC002516- 1376 nuoD 2984589- 2984589- 2984954_218_239_F 2984954_301_326_R 2961 PPS_NC002516- 311 PPS_NC002516- 1014 pps 1915014- 1915014- 1915383_44_63_F 1915383_140_165_R 2962 PPS_NC002516- 365 PPS_NC002516- 1052 pps 1915014- 1915014- 1915383_240_258_F 1915383_341_360_R 2963 TRP_NC002516- 527 TRP_NC002516- 1071 trpE 671831- 671831- 672273_24_42_F 672273_131_150_R 2964 TRP_NC002516- 490 TRP_NC002516- 1182 trpE 671831- 671831- 672273_261_282_F 672273_362_383_R

It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment.

Example 18 Selection and Use of Primer Pairs for Identification of Species of Bacteria Involved in Sepsis

In this example, identification of bacteria known to cause sepsis was accomplished using a panel of primer pairs chosen specifically with the aim of identifying these bacteria. The primer pairs of Table 25 were initially listed in Table 2. Additionally, primer pair numbers 346, 348, 349, 354, 358, 359, and 449 were listed in Table 5, as members of a bacterial surveillance panel. In this current example, the more specific group of bacteria known to be involved in causing sepsis is to be surveyed, Therefore, in development of this current panel of primer pairs, the surveillance panel of Table 5 has been reduced and an additional primer pair, primer pair number 2295 has been added. The primer members of primer pair 2295 hybridize to the tufB gene and produce a bioagent identifying amplicon for members of the family Staphylococcaceae which includes the genus Staphylococcus.

TABLE 25 Primer Pair Panel for Characterization of Septicemia Pathogens Forward Primer Reverse Primer (SEQ Primer Pair ID Reverse Primer (SEQ ID Target No. Forward Primer Name NO:) Name NO:) Gene 346 16S_EC_713_732_TMOD_F 202 16S_EC_789_809_TMOD_R 1110 16S rRNA 348 16S_EC_785_806_TMOD_F 560 16S_EC_880_897_TMOD_R 1278 16S rRNA 349 23S_EC_1826_1843_TMOD_F 401 23S_EC_1906_1924_TMOD_R 1156 23S rRNA 354 RPOC_EC_2218_2241_TMOD_F 405 RPOC_EC_2313_2337_TMOD_R 1072 rpoC 358 VALS_EC_1105_1124_TMOD_F 385 VALS_EC_1195_1218_TMOD_R 1093 valS 359 RPOB_EC_1845_1866_TMOD_F 659 RPOB_EC_1909_1929_TMOD_R 1250 rpoB 449 RPLB_EC_690_710_F 309 RPLB_EC_737_758_R 1336 rplB 2249 TUFB_NC002758- 430 TUFB_NC002758- 1321 tufB 615038- 615038- 616222_696_725_F 616222_793_820_R

To test for potential interference of human DNA with the present assay, varying amounts of bacterial DNA from E. coli 0157 and E. coli K-12 were spiked into samples of human DNA at various concentration levels. Amplification was carried out using primer pairs 346, 348, 349, 354, 358 and 359 and the amplified samples were subjected to gel electrophoresis. Smearing was absent on the gel, indicating that the primer pairs are specific for amplification of the bacterial DNA and that performance of the primer pairs is not appreciably affected in the presence of high levels of human DNA such as would be expected in blood samples. Measurement of the amplification products indicated that E. coli 0157 could be distinguished from E. coli K-12 by the base compositions of amplification products of primer pairs 358 and 359. This is a useful result because E. coli 0157 is a sepsis pathogen and because E. coli K-12 is a low-level contaminant of the commercially obtained Taq polymerase used for the amplification reactions.

A test of 9 blinded mixture samples was conducted as an experiment designed to simulate a potential clinical situation where bacteria introduced via skin or oral flora contamination could confound the detection of sepsis pathogens. The samples contained mixtures of sepsis-relevant bacteria at different concentrations, whose identities were not known prior to measurements. Tables 26A and 26B show the results of the observed base compositions of the amplification products produced by the primer pairs of Table 25 which were used to identify the bacteria in each sample. Without prior knowledge of the bacteria included in the 9 samples provided, it was found that samples 1-5 contained Proteus mirabilis, Staphylococcus aureus, and Streptococcus pneumoniae at variable concentration levels as indicated in Tables 26A and 26B. Sample 6 contained only Staphylococcus aureus. Sample 7 contained only Streptococcus pneumoniae. Sample 8 contained only Proteus mirabilis. Sample 9 was blank.

Quantitation of the three species of bacteria was carried out using calibration polynucleotides as described herein. The levels of each bacterium quantitated for each sample was found to be consistent with the levels expected.

This example indicates that the panel of primer pairs indicated in Table 25 is useful for identification of bacteria that cause sepsis.

In another experiment, two blinded samples were provided, The first sample, labeled “Germ A” contained Enterococcus faecalis and the second sample, labeled “Germ B” contained other Klebsiella pneumoniae. For “Germ A” the panel of primer pairs of Table 25 produced four bioagent identifying amplicons from bacterial DNA and primer pair numbers 347, 348, 349 and 449 whose base compositions indicated the identity of “Germ A” as Enterococcus faecalis. For “Germ B” the panel of primer pairs of Table 25 produced six bioagent identifying amplicons from bacterial DNA and primer pair numbers 347, 348, 349, 358, 359 and 354 whose base compositions indicated the identity of “Germ B” as Klebsiella pneumoniae.

One with ordinary skill in the art will recognize that one or more of the primer pairs of Table 25 could be replaced with one or more different primer pairs from Table 2 should the analysis require modification such that it would benefit from additional bioagent identifying amplicons that provide bacterial identification resolution for different species of bacteria and strains thereof.

TABLE 26A Observed Base Compositions of Blinded Samples of Amplification Products Produced with Primer Pair Nos. 346, 348, 349 and 449 Organism Organism Concentration Primer Pair Primer Pair Primer Pair Primer Pair Sample Component (genome copies) Number 346 Number 348 Number 349 No. 449 1 Proteus 470 A29G32C25T13 — — — mirabilis 1 Staphylococcus >1000 — A30G29C30T29 A26G3C25T20 — aureus 1 Streptococcus >1000 — A26G32C28T30 A28G31C22T20 A22G20C19T14 pneumoniae 2 Staphylococcus >1000 A27G30C21T21 A30G29C30T29 A26G30C25T20 — aureus 2 Streptococcus >1000 — — — A22G20C19T14 pneumoniae 2 Proteus 390 — — — — mirabilis 3 Proteus >10000 A29G32C25T13 A29G30C28T29 A25G31C27T20 — mirabilis 3 Streptococcus 675 — — — A22G20C19T14 pneumoniae 3 Staphylococcus 110 — — — — aureus 4 Proteus 2130 A29G32C25T13 A29G30C28T29 A25G31C27T20 — mirabilis 4 Streptococcus >3000 — A26G32C28T30 A28G31C22T20 A22G20C19T14 pneumoniae 4 Staphylococcus 335 — — — — aureus 5 Proteus >10000 A29G32C25T13 A29G30C28T29 A25G31C27T20 — mirabilis 5 Streptococcus 77 — — — A22G20C19T14 pneumoniae 5 Staphylococcus >1000 aureus 6 Staphylococcus 266 A27G30C21T21 A30G29C30T29 A26G30C25T20 — aureus 6 Streptococcus 0 — — — pneumoniae 6 Proteus 0 — — — — mirabilis 7 Streptococcus 125 — A26G32C28T30 A28G31C22T20 A22G20C19T14 pneumoniae 7 Staphylococcus 0 — — — — aureus 7 Proteus 0 — — — — mirabilis 8 Proteus 240 A29G32C25T13 A29G30C28T29 A25G31C27T20 — mirabilis 8 Streptococcus 0 — — — — pneumoniae 8 Staphylococcus 0 — — — — aureus 9 Proteus 0 — — — — mirabilis 9 Streptococcus 0 — — — — pneumoniae 9 Staphylococcus 0 — — — — aureus

TABLE 26B Observed Base Compositions of Blinded Samples of Amplification Products Produced with Primer Pair Nos. 358, 359, 354 and 2249 Organism Organism Concentration Primer Pair Primer Pair Primer Pair Primer Pair Sample Component (genome copies) Number 358 Number 359 Number 354 No. 2249 1 Proteus 470 — — A29G29C35T29 — mirabilis 1 Staphylococcus >1000 — — A30G27C30T35 A43G28C19T35 aureus 1 Streptococcus >1000 — — — — pneumoniae 2 Staphylococcus >1000 — — A30G27C30T35 A43G28C19T35 aureus 2 Streptococcus >1000 — — — — pneumoniae 2 Proteus 390 — — A29G29C35T29 — mirabilis 3 Proteus >10000 — — A29G29C35T29 — mirabilis 3 Streptococcus 675 — — — — pneumoniae 3 Staphylococcus 110 — — — A43G28C19T35 aureus 4 Proteus 2130 — — A29G29C35T29 — mirabilis 4 Streptococcus >3000 — — — — pneumoniae 4 Staphylococcus 335 — — — A43G28C19T35 aureus 5 Proteus >10000 — — A29G29C35T29 — mirabilis 5 Streptococcus 77 — — — — pneumoniae 5 Staphylococcus >1000 — — — A43G28C19T35 aureus 6 Staphylococcus 266 — — — A43G28C19T35 aureus 6 Streptococcus 0 — — — — pneumoniae 6 Proteus 0 — — — — mirabilis 7 Streptococcus 125 — — — — pneumoniae 7 Staphylococcus 0 — — — — aureus 7 Proteus 0 — — — — mirabilis 8 Proteus 240 — — A29G29C35T29 — mirabilis 8 Streptococcus 0 — — — — pneumoniae 8 Staphylococcus 0 — — — — aureus 9 Proteus 0 — — — — mirabilis 9 Streptococcus 0 — — — — pneumoniae 9 Staphylococcus 0 — — — — aureus

Example 19 Design and Validation of Primer Pairs Designed for Production of Amplification Products from DNA of Sepsis-Causing Bacteria

The following primer pair numbers were designed to provide an improved collection of bioagent identifying amplicons for the purpose of identifying sepsis-causing bacteria: 3346 (SEQ ID NOs: 1448:1461), 3347 (SEQ ID NOs: 1448:1464), 3348 (SEQ ID NOs: 1451:1464), 3349 (SEQ ID NOs: 1450:1463), 3350 (SEQ ID NOs: 309:1458), 3351 (SEQ ID NOs: 309:1460), 3352 (SEQ ID NOs: 1445:1458), 3353 (SEQ ID NOs: 1447:1460), 3354 (SEQ ID NOs: 309:1459), 3355 (SEQ ID NOs: 1446:1458), 3356 (SEQ ID NOs: 1452:1467), 3357 (SEQ ID NOs: 1452:1465), 3358 (SEQ ID NOs: 1453:1466), 3359 (SEQ ID NOs: 1449:1462), 3360 (SEQ ID NOs: 1444:14570), 3361 (SEQ ID NOs: 1454:1468), 3362 (SEQ ID NOs: 1455:1469), and 3363 (SEQ ID NOs: 1456:1470).

Primer pair numbers 3346-3349, and 3356-3359 have forward and reverse primers that hybridize to the rpoB gene of sepsis-causing bacteria. The reference gene sequence used in design of these primer pairs is an extraction of nucleotide residues 4179268 to 4183296 from the genomic sequence of E. coli K12 (GenBank Accession No. NC_(—)000913.2, gi number 49175990). All coordinates indicated in the primer names are with respect to this sequence extraction. For example, the forward primer of primer pair number 3346 is named RPOB_NC000913_(—)3704_(—)3731_F (SEQ ID NO: 1448). This primer hybridizes to positions 3704 to 3731 of the extraction or positions 4182972 to 4182999 of the genomic sequence. Of this group of primer pairs, primer pair numbers 3346-3349 were designed to preferably hybridize to the rpoB gene of sepsis-causing gamma proteobacteria. Primer pairs 3356 and 3357 were designed to preferably hybridize to the rpoB gene of sepsis-causing beta proteobacteria, including members of the genus Neisseria. Primer pairs 3358 and 3359 were designed to preferably hybridize to the rpoB gene of Corynebacteria and Mycobacteria.

Primer pair numbers 3350-3355 have forward and reverse primers that hybridize to the rplB gene of gram positive sepsis-causing bacteria. The forward primer of primer pair numbers 3350, 3351 and 3354 is RPLB_EC_(—)690_(—)710_F (SEQ ID NO: 309). This forward primer had been previously designed to hybridize to GenBank Accession No. NC_(—)000913.1, gi number 16127994 (see primer name code RPLB_EC in Table 3). The reference gene sequence used in design of the remaining primers of primer pair numbers 3350-3355 is the reverse complement of an extraction of nucleotide residues 3448565 to 3449386 from the genomic sequence of E. coli K12 (GenBank Accession No. NC_(—)000913.2, gi number 49175990). All coordinates indicated in the primer names are with respect to the reverse complement of this sequence extraction. For example, the forward primer of primer pair number 3352 is named RPLB_NC000913_(—)674_(—)698_F (SEQ ID NO: 1445). This primer hybridizes to positions 674-698 of the reverse complement of the extraction or positions 3449239 to 3449263 of the reverse complement of the genomic sequence. This primer pair design example demonstrates that it may be useful to prepare new combinations of primer pairs using previously existing forward or reverse primers.

Primer pair number 3360 has a forward primer and a reverse primer that both hybridize to the gyrB gene of sepsis-causing bacteria, preferably members of the genus Streptococcus. The reference gene sequence used in design of these primer pairs is an extraction of nucleotide residues 581680 to 583632 from the genomic sequence of Streptococcus pyogenes M1 GAS (GenBank Accession No. NC_(—)002737.1, gi number 15674250). All coordinates indicated in the primer names are with respect to this sequence extraction. For example, the forward primer of primer pair number 3360 is named GYRB_NC002737_(—)852_(—)879_F (SEQ ID NO: 1444). This primer hybridizes to positions 852 to 879 of the extraction.

Primer pair number 3361 has a forward primer and a reverse primer that both hybridize to the tufB gene of sepsis-causing bacteria, preferably gram positive bacteria. The reference gene sequence used in design of these primer pairs is an extraction of nucleotide residues 615036 . . . 616220 from the genomic sequence of Staphylococcus aureus subsp. aureus Mu50 (GenBank Accession No. NC_(—)002758.2, gi number 57634611). All coordinates indicated in the primer names are with respect to this sequence extraction. For example, the forward primer of primer pair number 3360 is named TUFB_NC002758_(—)275_(—)298_F (SEQ ID NO: 1454). This primer hybridizes to positions 275 to 298 of the extraction.

Primer pair numbers 3362 and 3363 have forward and reverse primers that hybridize to the valS gene of sepsis-causing bacteria, preferably including Klebsiella pneumoniae and strains thereof. The reference gene sequence used in design of these primer pairs is the reverse complement of an extraction of nucleotide residues 4479005 to 4481860 from the genomic sequence of E. coli K12 (GenBank Accession No. NC_(—)000913.2, gi number 49175990). All coordinates indicated in the primer names are with respect to the reverse complement of this sequence extraction. For example, the forward primer of primer pair number 3362 is named VALS_NC000913_(—)1098_(—)1115_F (SEQ ID NO: 1455). This primer hybridizes to positions 1098 to 1115 of the reverse complement of the extraction.

In a validation experiment, samples containing known quantities of known sepsis-causing bacteria were prepared. Total DNA was extracted and purified in the samples and subjected to amplification by PCR according to Example 2 and using the primer pairs described in this example. The three sepsis-causing bacteria chosen for this experiment were Enterococcus faecalis, Klebsiella pneumoniae, and Staphylococcus aureus. Following amplification, samples of the amplified mixture were purified by the method described in Example 3 subjected to molecular mass and base composition analysis as described in Example 4.

Amplification products corresponding to bioagent identifying amplicons for Enterococcus faecalis were expected for primer pair numbers 3346-3355, 3360 and 3361. Amplification products were obtained and detected for all of these primer pairs.

Amplification products corresponding to bioagent identifying amplicons for Klebsiella pneumoniae were expected and detected for primer pair numbers 3346-3349, 3356, 3358, 3359, 3362 and 3363. Amplification products corresponding to bioagent identifying amplicons for Klebsiella pneumoniae were detected for primer pair numbers 3346-3349 and 3358.

Amplification products corresponding to bioagent identifying amplicons for Staphylococcus aureus were expected and detected for primer pair numbers 3348, 3350-3355, 3360, and 3361. Amplification products corresponding to bioagent identifying amplicons for Klebsiella pneumoniae were detected for primer pair numbers 3350-3355 and 3361.

CONCLUDING STATEMENTS

The present invention includes any combination of the various species and subgeneric groupings falling within the generic disclosure. This invention therefore includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

While in accordance with the patent statutes, description of the various embodiments and examples have been provided, the scope of the invention is not to be limited thereto or thereby. Modifications and alterations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.

Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific examples which have been presented by way of example.

Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank gi or accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety. 

1. A method for identifying a sepsis-causing bacterium in a sample, comprising: a) amplifying a nucleic acid from said sample using an oligonucleotide primer pair comprising a forward primer and a reverse primer, said forward primer comprising 21-35 linked nucleotides and comprising the sequence of SEQ ID NO: 309, and said reverse primer comprising 24-35 linked nucleotides and comprising the sequence of SEQ ID NO: 1458; and b) determining the molecular mass of said amplification product by mass spectrometry.
 2. The method of claim 1, further comprising comparing said molecular mass to a database comprising a plurality of molecular masses of bioagent identifying amplicons, wherein a match between said determined molecular mass and a molecular mass in said database identifies said sepsis-causing bacterium in said sample.
 3. The method of claim 1, further comprising calculating a base composition of said amplification product using said molecular mass.
 4. The method of claim 3, further comprising comparing said calculated base composition to a database comprising a plurality of base compositions of bioagent identifying amplicons, wherein a match between said calculated base composition and a base composition included in said database identifies said sepsis-causing bacterium in said sample.
 5. The method of claim 1, further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair wherein the primers of each of said at least one additional primer pair are designed to hybridize to a bacterial gene selected from the group consisting of 16S rRNA, 23S rRNA, tufB, rpoB, valS, rplB, and gyrB.
 6. The method of claim 1, wherein said molecular mass identifies the presence of said sepsis-causing bacterium in said sample.
 7. The method of claim 6, further comprising determining either sensitivity or resistance of said sepsis-causing bacterium in said sample to one or more antibiotics.
 8. The method of claim 1, wherein said molecular mass identifies a sub-species characteristic, strain, or genotype of said sepsis-causing bacterium in said sample.
 9. A method for identifying at least one sepsis causing bacteria from a sample comprising the steps of: a) obtaining a sample; b) contacting at least one nucleic acid from said sample with at least one purified oligonucleotide primer pair from claim 1; c) performing an amplification reaction, thereby generating at least one amplification product and; d) analyzing at least one amplification product from step c to identify at least one sepsis causing bacteria in said sample.
 10. The method of claim 9 wherein said analyzing step is selected from the group consisting of mass spectrometry analysis, real time PCR analysis, sequencing analysis, hybridization analysis, hybridization protection assay analysis and mass array analysis.
 11. The method of claim 10 wherein said mass spectrometry analysis is ESI TOF mass spectrometry.
 12. The method of claim 10 wherein said mass spectrometry analysis comprises generating molecular mass data for said amplification product.
 13. The method of claim 12 further comprising calculating a base composition from said generated molecular mass data.
 14. The method of claim 12 further comprising comparing said molecular mass data to a plurality of molecular masses in a database, wherein said plurality of molecular masses are indexed to said oligonucleotide primer pairs and to a plurality of known sepsis causing bacteria, and wherein a match between said generated molecular mass data and a member of said plurality of molecular masses identifies at least one sepsis causing bacteria in said sample.
 15. The method of claim 13 further comprising comparing said base composition to a plurality of base compositions in a database, wherein said plurality of base compositions are indexed to said primer pairs and to a plurality of known sepsis causing bacteria, and wherein a match between said base composition and a member of said plurality of base compositions identifies at least one sepsis causing bacteria in said sample.
 16. The method of claim 14 wherein said at least one sepsis causing bacteria is identified by genus, species, sub-species, serotype or genotype.
 17. The method of claim 15 wherein said at least one sepsis causing bacteria is identified by genus, species, sub-species, serotype or genotype.
 18. The method of claim 10 wherein said at least one amplification product in step d is substantially purified before analysis.
 19. The method of claim 18 wherein said at least one amplification product is substantially purified using a magnetic bead covalently linked with an ion exchange resin. 